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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  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.  ii  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.  iii  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   iv  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   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   3  Samples ............................................................................................................................... 16   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   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   v  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  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   4  Comparison of copy number altered genes between RB1+/+, RB1+/-, and RB1-/- tumors . 40   MYCNA and age of diagnosis .............................................................................................. 54   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   vi  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   vii  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   viii  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 ix  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 x  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  xi  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.  xii  Dedication This thesis is dedicated to my family and friends.  xiii  1 Chapter: Introduction 1.1  1.1.1  Retinoblastoma  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).  1  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 2  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  1.2.1  Retinoblastoma genetics  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 3  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).  4  pRB  P  Phosphorylation by Cyclin/ cdk  complexes  E2F DP pRB  Transient process during cell  cycle progression E2F  DP  DNA E2F target gene transcription  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 WeriRb-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 5  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 knockout 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  6  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 7  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 8  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. 9  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 microtubuledependent 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.  10  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 11  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; 12  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.  13  1.3  1.3.1  Hypotheses and research questions  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+/+ 14  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.  15  2 Chapter: Materials and Methods 2.1  2.1.1  Samples  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 QmPCR 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 allelespecific 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). 16  Incidence of RB1+/+ retinoblastoma across the four testing sites  2.1.3  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 Submegabase 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.  17  1. DNA Labelling  5. Visualization Cell Line Karyogram 0  1p36.33  Focal amplifications  Differentially labelled sample and reference DNA 1p31.2 1p13.3  2. Competitive Co-hybridization  1q12  Gain Magnified view of a single grid of array elements spotted in duplicate (ex. BAC clones)  3. Washing and 4. Imaging  1q32.1  Loss 1q44  Chr. 1 Normalized log2 signal intensity ratio plotted against chromosomal position for each array element  6. Data Analysis Background normalization, breakpoint detection, alteration calling and comparisons across multiple samples  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 18  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 19  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.  20  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 21  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 nonMYCNA 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 22  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 righttailed 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  2.3.1  Affymetrix genome-wide SNP 6.0 array Genotyping  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). 23  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.  24  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.  25  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 26  (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. 27  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.  28  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 29  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). 30  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.  31  -.5  .5  -.5  .5 -.5  .5  -.5  .5  -.5  .5  -.5  .5 -.5  -.5  .5  -.5  .5  -.5  10  9  -.5  3  2  1  .5  -.5  17  .5  -.5  .5  -.5  .5  19  5  .5  -.5  12  11  18  Figure 3.5  4  -.5  -.5  .5  21  .5  14  13  .5  20  -.5  -.5  -.5  7  6  .5  .5  -.5  .5  8  .5  -.5  15  .5  16  .5  22  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 32  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  33  chromosome 2p from bps 15998134 to 16004580), is the only gene contained within the minimal region of amplification.  DDX1 NAG  2p24.3  2p25.1  RefSeq  (NCBI36/hg18)  Genes  MYCN  Figure 3.6  Y79 C1  P5  RB522  E26  P2 E7 E4  T42  T25  T5 T14  T33 RB381  2p24.1  2p24.2  FAM49A  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.  34  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).  35  Table 3.1 All discreet high level amplicons identified. Status  Sample  Chr  Start  Start (bp)  End Clone  End (bp)  Clone RB1+/+  T5  T42  Size  # of  Known  (Mb)  genes  Oncogenes  1q41  N0282E08  217613871  N0514C19  219396428  1.78  10  2p24.3  N0451A14  15703698  N0065N17  16825910  1.12  2  21q22.3  M2410F14  44478602  M2053G20  44754475  0.28  8  2p25.1  N0487B06  11436647  N0641J22  11754223  0.32  3  2p24.3-  N0571E19  14565626  N0427M01  17376543  2.81  5  MYCN  MYCN  2p24.2 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-  N0619O15  15175256  N0325P07  17748109  2.57  6  MYCN  N0619O15  15175256  N0631D03  17177195  2.00  4  MYCN  N0686G09  9935380  N0062M03  16432161  6.50  26  MYCN  N0541K19  15467410  N0554B24  18350885  2.88  8  MYCN  N0196C08  13032063  N1007E02  17123259  4.09  5  MYCN  N0723F23  14978618  M2305P22  18851300  3.87  9  MYCN  N0091E09  14051860  N0554B24  18350885  4.30  8  MYCN  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  2p25.2-  N0129I01  5022521  N0517E08  9267587  4.25  8  N0541K19  15467410  N0419K22  18088207  2.62  8  2p24.2 E7  2p24.32p24.2  P2  2p25.12p24.3  P5  2p24.32p24.2  E26  2p24.32p24.2  C1  2p24.32p24.2  RB522  2p24.32p24.2  RB1  -/-  RB2052  RB381  GPC5  2p25.1 2p24.32p24.2  36  MYCN  Status  Sample  RB1-/-  Chr  T23  Start Clone  12q24.31-  Start (bp)  End Clone  End (bp)  Size  # of  Known  (Mb)  genes  Oncogenes  N0387F15  122580551  N0006I24  125366514  2.79  18  12q24.32  N0016J23  125921569  N0337L12  128699668  2.78  3  1q32.1  N0783D13  201524037  N0421E17  204708300  3.18  50  MDM4  N9463P22  16001272  N1007E02  17123259  1.12  2  MYCN  12q24.32  E27  *13q RB1+/-  T33  2p24.32p24.2  *Tumor E27 contains many rearrangements including too many high level amplification events on chromosome 13q to list  A  1q31.3 1q32.1 1q32.2 1q32.3  50 Genes: including MDM4  1q41 1q42.11  RB2052  B  T5*  E27  Genes: SLC30A10, EPRS, BPNT1, IARS2, RAB3GAP2, MARK1, C1orf115, MOSC2, MOSC1, HLX1  C Genes: E2F6, GREB1, NTSR2  2p25.1 2p24.3 2p24.2  21q22.3  2p21  T5*  Genes: ICOSLG, DNMT3L, AIRE, PFKL, C21orf2, TRPM2, LRRC3, C21orf29, KRTAP10-1, 2, -3, -5, -6, -9, -10, -11, KRTAP12-3, -2, -1, UBE2G2  2p13.3 2p13.1 2p12  T42  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, 37  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).  38  A. Comparison of the number of alterations between tumor groups  Number of Alterations  (p = 0.1335; unpaired t-test)  Number of base pairs altered  B. Comparison of the number of bps altered between tumor groups  (p = 0.0109; unpaired t-test)  39  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). Comparison of copy number altered genes between RB1+/+, RB1+/-, and RB1-/-  3.5.1 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.  40  RB1‐/‐  RB1+/+ Without MYCNA 292 1285   1037 109  227  151 763  RB1+/+ MYCNA  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  3.6.1  Pathway/ network level analysis  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 41  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.  42  A A A A  Figure 3.10  B B B B  Binding only Acts on Inhibits Inhibits and acts on  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.  43  A A A A  Figure 3.11  B B B B  Binding only Acts on Inhibits Inhibits and acts on  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.  44  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.  45  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  T45  7  123528211  150253930  35010  2.92  11  77729342  122914228  45185  2.90  14  105472642  105818073  345  3.50  2  47047732  47126711  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  Deletion  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  2  2784  11597833  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  3013  1.09  2  17445973  20999949  3554  1.10  2  224189680  242738130  18548  1.43  X  10452961  12583078  2130  1.27  46  Amplification  Deletion  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  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  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  2  15706592  18017505  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  T5  T14  Deletion  Amplification  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  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  47  Deletion  *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.  48  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  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  2  2784  11597833  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  3013  1.09  2  17445973  20999949  3554  1.1  2  224189680  242738130  18548  1.43  X  10452961  12583078  2130  1.27  T45  Deletion  Amplification  Deletion  49  Sample T25  T5  T14  Chromosome 2  Start (bp) 14568303  End (bp) 16749334  7  38258932  7  Copy Number Status Amplification  Length (kb) 2181  Mean* 17.9  38318607  60  2.62  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  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 2 7 7 11  21500741 69401585 410 13087736 14653556  22029795 69760144 16150 14653556 15682120  529 359 16 1566 1029  2.62 2.51 2.76 1.06 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  2 4  15706592 170175614  18017505 191261905  2311 21056  14.47 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 6  410 65436733  16150 65497247  16 61  2.62 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  Deletion  Amplification  Deletion  50  *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  51  40 30  T25  Minor alleles  RB522 T5  T45 T14  T25  RB522 T5  T45 T14  T25  10  20  T45 T14  20 10 0  Copy number  Total copy number  30  40  0  Copy number  RB522 T5  Allele specific copy number  Major alleles  2p25.1  2p24.3  2p24.2  52  2p24.1  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 53  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.  54  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+/+ 55  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, 56  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  57  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 58  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  4.8.1  Conclusions  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 59  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. 60  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. 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"Mechanisms of loss of heterozygosity in retinoblastoma." Cytogenet Cell Genet 59(4): 248-252. Zielinski, B., S. Gratias, et al. (2005). "Detection of chromosomal imbalances in retinoblastoma by matrix-based comparative genomic hybridization." Genes Chromosomes Cancer 43(3): 294-301.  72  Appendices Appendix A Additional aCGH data  A.1  Genomic regions of gain (G) and loss (L) identified by aCGH for all 48 samples  and two cell lines Table A.1 shows all copy number alterations of two or more array elements identified in each of the 48 primary tumors and two cell lines. Figures A1 and A2 show the aCGH karyograms for each cell line profiled.  Table A.1  aCGH alterations by sample (RB1+/+).  ID  G/L  Chr  Start (bp)  End (bp)  E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4  G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G  1 1 1 1 2 2 2 3 3 5 6 7 7 7 7 7 7 7 7 9 9 11 11 12 14 14 14 14 14 15  16646685 44122639 88987745 232727671 14978618 24892640 121240000 49081051 198661730 125838199 158979778 97475547 100027063 105069977 127398355 127848914 131677611 141206417 148316114 45908108 91235105 61823795 67511350 2717303 73121067 94620036 96434336 98532350 104222750 30328715  17141712 44370880 89217390 233010148 17715047 25572843 122362789 49782857 198819853 126214785 159379225 98965520 102036700 105602595 127812452 129934305 132059225 142434257 149799209 46284566 91864006 62111938 68042929 2975966 73575089 95405991 97482889 103624847 104589570 30881934  Size (kb) 495 248 230 282 2736 680 1123 702 158 377 399 1490 2010 533 414 2085 382 1228 1483 376 629 288 532 259 454 786 1049 5092 367 553  73  ID E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19  G/L G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G  Chr  Start (bp)  End (bp)  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 2 2 2 2 2  16646685 52091543 88444733 142533622 149858970 152795955 158010337 159680000 162887424 164082233 173228930 174639051 179071822 182037344 199215670 199831132 204118116 208781386 214852240 218273469 219082723 222753539 226230204 232727671 235641020 41662 1642610 3903894 12063690 25273765  17086056 52349308 88711100 144288841 151131174 153230241 158996174 160677252 163728359 165430178 174094669 175287621 180163431 182693973 199653218 203411834 208088220 209169786 215267049 218795330 220169719 223064465 228803178 233105801 236172564 1218689 3489907 11755093 23881796 26864767  Size (kb) 439 258 266 1755 1272 434 986 997 841 1348 866 649 1092 657 438 3581 3970 388 415 522 1087 311 2573 378 532 1177 1847 7851 11818 1591  ID  G/L  Chr  Start (bp)  End (bp)  E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4  G G G G G G G G G G G G G G G L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L  15 15 16 16 16 16 17 17 17 17 20 20 21 21 22 1 2 2 2 2 3 3 3 5 5 6 6 6 6 6 6 6 6 6 7 7 7 7 7 7 7 7 8 8 8 8 8  72441859 76894500 30539947 33207279 82567035 88443716 510963 37016754 56678248 69307909 54443015 60809452 20794703 43761584 21035693 24642063 76560762 91608685 109113179 158728287 26243462 100412584 190572982 49479231 115399904 58352741 62000083 64287458 77454026 86423400 93274524 95517378 113443711 160616018 9862060 11969246 18699485 57765880 61061239 66502015 87945541 127648906 16092289 43185803 86699540 132297428 143780694  73217136 77184857 31120743 33463485 82963492 88573088 1628597 37707027 57089085 69931286 54747849 61325197 21052440 44194069 21445636 24852567 77046290 91680249 109402865 159062951 26651493 100932125 190793756 49913062 115637029 58849697 63102316 65557020 77857011 87841029 93823839 96361943 113802827 161039688 10928986 12542008 20220381 58017280 62176519 66754917 88499287 127924442 16348466 43912178 87055819 132772696 144006276  Size (kb) 775 290 581 256 396 129 1118 690 411 623 305 516 258 432 410 211 486 72 290 335 408 520 221 434 237 497 1102 1270 403 1418 549 845 359 424 1067 573 1521 251 1115 253 554 276 256 726 356 475 226  74  ID E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19 E19  G/L  Chr  Start (bp)  End (bp)  G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G L L L L L L L L  2 2 2 2 2 2 2 2 4 4 4 5 5 5 5 6 6 7 7 10 10 10 11 12 12 12 12 12 12 13 13 13 13 14 15 15 15 18 22 3 6 7 11 11 15 16 16  27134515 44824074 53351511 54256708 56202060 63797391 70684434 83467797 3645088 67582165 109386058 57285917 85900497 95697117 118943200 73242 45494917 57163665 83422963 49935163 87854528 97978547 60661723 51140849 52090606 66859847 108154779 114113748 121949281 43581707 55372389 108629457 110600643 93699417 21973970 28106102 77142292 42657928 30095758 78394977 160616018 1542959 97278670 125166505 19842977 20943307 33730527  43220834 52869360 53831046 55105184 62301010 70677309 82714465 89405301 4065877 67861860 109668156 57537297 86163409 95852946 119294214 45175251 58849697 57368235 83649776 50237413 88147135 98240450 60880212 51492452 52327387 67063558 108581445 114520437 122155980 43926080 55655559 109193347 112590314 94182499 22386839 28734907 77855965 42803873 30360524 78583892 161039688 1737551 97609941 125470718 20071155 21212616 34016785  Size (kb) 16086 8045 480 848 6099 6880 12030 5938 421 280 282 251 263 156 351 45102 13355 205 227 302 293 262 218 352 237 204 427 407 207 344 283 564 1990 483 413 629 714 146 265 189 424 195 331 304 228 269 286  ID  G/L  Chr  Start (bp)  End (bp)  E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E4 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7  L L L L L L L L L L L L L L L G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G  9 9 9 9 10 10 11 11 11 12 12 13 16 17 19 1 1 1 2 2 3 3 3 4 6 6 6 6 6 6 6 6 6 6 6 6 8 9 9 10 11 12 12 12 13 14 14  67812022 90861578 105583846 129150841 41947396 113323636 38072737 58901926 97278670 36302222 77277923 73742673 33659117 7088883 50398622 1058909 21717568 232830875 15182883 64982297 121682380 137801269 198661730 189018797 213736 4800956 6705438 10144863 23259241 25059720 30758621 32872724 36804059 39096919 39928065 158979778 58157391 132643829 136245273 69245834 47710156 48413812 54361649 97265837 113932258 68087188 89221786  68761941 91085013 105969389 129545178 42224061 113623272 38323517 59283911 97609941 36786132 77556391 74123958 34016785 7457682 50652469 2545127 22285488 233010148 17177195 65322374 121964439 138197862 198819853 189277190 1923096 5673594 7834700 11419717 23609362 26850182 32151955 36488793 38315288 39555660 44057109 159379225 58393591 132999756 136359804 69639428 48122594 49295862 54722693 97621020 114093192 68444627 90956790  Size (kb) 950 223 386 394 277 300 251 382 331 484 278 381 358 369 254 1486 568 179 1994 340 282 397 158 258 1709 873 1129 1275 350 1790 1393 3616 1511 459 4129 399 236 356 115 394 412 882 361 355 161 357 1735  75  ID E19 E19 E22 E22 E22 E22 E22 E22 E22 E22 E22 E22 E22 E22 E22 E22 E22 E22 E22 E22 E22 E22 E22 E22 E22 E22 E22 E22 E22 E22 E22 E22 E22 E22 E22 E22 E22 E22 C2 C2 C2 C2 C2 C2 C2 C2 C2  G/L  Chr  Start (bp)  End (bp)  L L G G G G G G G G G G G G G G G G G G G L L L L L L L L L L L L L L L L L G G G G G G G G G  18 22 1 1 3 4 4 5 5 5 6 7 7 8 9 15 15 15 16 19 20 5 6 6 7 7 7 7 11 11 12 14 14 16 19 21 22 22 1 1 1 1 1 1 1 1 1  16659756 40739833 143665914 157111728 141103332 134269189 190667654 568397 17284044 71207958 53635140 57163665 141707706 58157391 122056354 21973970 28106102 30237319 33280312 47784067 44119293 127204007 13223270 74106063 1542959 61087267 64729861 77228817 97278670 118808228 32108495 30416622 49100288 20943307 32504109 33637551 17053965 40935061 3519965 16646685 120249993 142533622 144594746 145511311 149858970 154247819 158010337  17277836 41198248 144162829 157249573 141471148 134522870 190733475 1013978 17553934 71428448 53820956 57368235 142316803 58393591 122361130 22386839 28734907 30597338 33741546 48626174 44472487 127468573 13524298 74476174 1737551 61270796 65014606 77535943 97609941 119073431 32334573 31059479 49484060 21212616 32613567 34149277 17206783 41198248 3748249 17086056 121077638 144162829 145133682 145872489 153230241 157619715 158996174  Size (kb) 618 458 497 138 368 254 66 446 270 220 186 205 609 236 305 413 629 360 461 842 353 265 301 370 195 184 285 307 331 265 226 643 384 269 109 512 153 263 228 439 828 1629 539 361 3371 3372 986  ID  G/L  Chr  Start (bp)  End (bp)  E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7 E7  G G G G G G G G G G G G G L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L  14 14 15 16 17 17 17 17 18 19 20 20 22 2 2 2 2 3 3 3 4 4 5 6 6 7 7 8 8 8 8 9 9 10 11 11 11 11 11 11 11 11 11 11 12 15 16  94620036 97186900 20302216 923071 623188 34574333 55870907 75566340 25034674 1331124 5506268 44981867 16245214 91303216 95774261 96783694 161249510 80918434 163846727 190572982 7507478 144726984 26139638 58352741 79956399 11969246 87945541 7035076 86699540 88151299 115440593 66254996 67803916 41947396 96953808 102841892 105035952 109773406 111537832 114281321 118950133 122460473 127686192 129706418 59227752 37191685 15040819  95405991 104589570 20627698 2131094 962070 34955272 56233848 75862414 25284594 2630211 5961085 45202369 16594536 91680249 96130285 96834810 161442145 81176295 164196155 190793756 7829767 144928389 26409556 58849697 80258421 12200051 88433327 8212432 87055819 88551969 115731250 67409630 70224724 42224061 97518886 103697609 106669861 110120305 113144857 117260839 121132876 127141998 128232929 134433506 59583586 37629456 15407666  Size (kb) 786 7403 325 1208 339 381 363 296 250 1299 455 221 349 377 356 51 193 258 349 221 322 201 270 497 302 231 488 1177 356 401 291 1155 2421 277 565 856 1634 347 1607 2980 2183 4682 547 4727 356 438 367  76  ID C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2  G/L  Chr  Start (bp)  End (bp)  G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G L L L L L L L L  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 3 3 4 5 5 5 7 8 8 11 11 13 15 15 15 15 17 19 3 3 4 6 7 7 11 12  159680000 161016469 162887424 168402600 173228930 174639051 177764373 179462543 182037344 195889657 196594358 199215670 212028920 218406885 222704527 223993446 234814970 237944944 242294867 91608685 113424819 52725023 114828518 134269189 568397 16358405 134308327 57163665 7035076 58157391 60661723 132051142 43481375 18473375 21973970 28106102 30237319 20185620 61714137 75285656 78394977 8979402 13223270 1499748 77228817 125166505 32108495  160677252 161777223 167445692 171187941 174094669 176344936 178479748 181045400 182693973 196341623 198833601 209169786 215373858 220609576 223152230 233391573 236172564 240324819 244296856 91680249 113704783 52967443 115117759 134522870 1013978 16598168 134619053 57368235 8212432 58393591 60880212 132389201 43926080 20053874 22386839 28734907 30597338 20448380 62000777 75515882 78583892 9291412 13524298 1737551 77535943 125470718 32334573  Size (kb) 997 761 4558 2785 866 1706 715 1583 657 452 2239 9954 3345 2203 448 9398 1358 2380 2002 72 280 242 289 254 446 240 311 205 1177 236 218 338 445 1580 413 629 360 263 287 230 189 312 301 238 307 304 226  ID  G/L  Chr  Start (bp)  End (bp)  E7 E7 E7 E7 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2  L L L L G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G  18 19 22 22 1 1 1 2 2 2 2 2 3 4 5 5 5 6 7 7 8 8 9 9 10 10 10 10 11 12 12 13 14 15 15 15 16 16 16 17 17 17 17 17 17 17 17  15026396 50398622 17053965 18401409 9725864 26950696 157848491 8123122 28278420 91608685 120629322 241955282 62915894 27244185 85900497 86432253 106184345 82918123 28368924 132216136 58157391 101972208 113559615 138368718 75055 28180758 69245834 73363600 118808228 54120030 62920426 113000386 23602843 19970520 28106102 82870937 1341140 70168706 80253332 623188 24695117 40215472 41755471 45294025 54544587 71250082 73871046  15320434 50652469 17206783 19036350 9983505 27234980 158603002 16432161 28756000 91680249 121861830 242720443 63393838 27690451 86206359 86737136 106509625 83251931 28712572 132737649 58393591 102297846 113848887 138503912 538407 28445060 69639428 74407034 119073431 54512007 63310916 113414382 23978395 20224003 28937648 83217528 2346105 70579581 80497048 962070 25127880 41357984 42728503 45685773 55060070 73457154 74851642  Size (kb) 294 254 153 635 258 284 755 8309 478 72 1233 765 478 446 306 305 325 334 344 522 236 326 289 135 463 264 394 1043 265 392 390 414 376 253 832 347 1005 411 244 339 433 1143 973 392 515 2207 981  77  ID C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2  G/L  Chr  Start (bp)  End (bp)  L L L L L L L L L L L L L L L G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G  12 16 16 16 16 16 16 16 16 16 16 18 19 19 22 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 5 5 6 6  53976376 28918888 47457492 50650921 52550335 60759769 64289064 69643219 71288486 75492204 79508959 25034674 4648531 32504109 17053965 617 9725864 17239175 36428818 145425752 149747994 153361602 158010337 176619795 182699897 199091008 205931802 223536766 228205983 241997888 245436183 925973 1831108 2815042 7369112 8295224 15598791 19690629 24892640 28214990 84979184 96074237 119880768 70263 172799092 5423679 30758621  54173283 29239140 50618041 51434198 56200067 63642008 67577655 70905058 73055634 78760401 88573088 25284594 4966988 32613567 17206783 3000997 9983505 19020943 37442774 145734554 150399374 155397482 158739744 176971757 183127629 203475222 206903358 224717804 229467946 242642629 245747506 1404470 2663054 3266649 7828054 11025497 16914958 21011770 25572843 28865200 86465542 96378723 121861830 1562826 173131927 5673594 32151955  Size (kb) 197 320 3161 783 3650 2882 3289 1262 1767 3268 9064 250 318 109 153 3000 258 1782 1014 309 651 2036 729 352 428 4384 972 1181 1262 645 311 478 832 452 459 2730 1316 1321 680 650 1486 304 1981 1493 333 250 1393  ID  G/L  Chr  Start (bp)  End (bp)  P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P5 P5 P5 P5 P5 P5 P5 P5 P5 P5 P5 P5 P5  G G G G G G G L L L L L L L L L L L L L L L L L L L L L L L L L L L G G G G G G G G G G G G G  17 19 19 19 21 22 22 1 1 2 3 3 5 6 8 8 8 8 8 8 8 8 8 9 9 10 10 10 16 17 19 19 19 20 1 1 1 1 2 3 5 6 6 8 14 14 15  75249971 3128263 5172606 17824690 46485627 37632987 39513597 74725261 180445938 159197634 30195993 44821716 62884026 160616018 304176 5294980 12725608 17799615 20179288 22768625 25235119 40646088 86699540 43012161 67322055 41947396 50596911 77487773 34750831 26147289 8934066 55439223 63182936 10711991 155943888 160338731 232727671 246424085 15467410 21893268 17284044 19267498 158979778 58157391 42674393 94478987 28106102  78615238 4966988 6694652 18030848 46927776 37982948 40036674 74916322 180878805 159555327 30479678 45416063 63168836 161039688 4307888 11933605 13280883 19423330 22320777 24986910 27192114 40940717 87055819 43277572 68460734 42224061 50869761 77592293 35136830 26497031 9026570 55707294 63654245 11003593 156178456 160677252 233105801 246944046 18682260 22110528 17553934 19549037 159379225 58393591 42924774 94732235 28734907  Size (kb) 3365 1839 1522 206 442 350 523 191 433 358 284 594 285 424 4004 6639 555 1624 2141 2218 1957 295 356 265 1139 277 273 105 386 350 93 268 471 292 235 339 378 520 3215 217 270 282 399 236 250 253 629  78  ID E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2  G/L  Chr  Start (bp)  End (bp)  G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G  6 6 6 6 6 6 7 7 7 7 7 7 8 8 9 9 9 10 10 11 11 11 12 12 13 13 13 14 15 15 15 15 15 15 16 16 16 17 17 17 17 17 17 17 17 18 19  32872724 39096919 40472908 139650310 157097294 167951793 2038803 20168369 97475547 155193677 156529437 158404469 28931963 144140067 129488250 132643829 136245273 132891454 133771689 47194406 64048806 67511350 122604516 130673016 28785074 32954484 110600643 103814279 27830649 30441504 62717015 71764685 76894500 98989163 44231 49932229 87260490 0 7516212 27213580 29749613 36547206 57842004 69307909 73330751 62103149 1331124  36862995 39555660 44268248 140003204 159379225 169148970 2407575 20427128 100193425 155735685 157364621 158818116 29117855 145740218 131906000 132999756 136359804 133271394 135065876 47629247 65814442 68042929 124234371 131701464 29182576 33795089 114093192 104995019 29601956 30966443 63392257 72141641 77640945 100309437 1111069 50295218 87542642 1628597 9162774 28028278 30490642 44892002 58351860 73230854 78615238 62501278 7667777  Size (kb) 3990 459 3795 353 2282 1197 369 259 2718 542 835 414 186 1600 2418 356 115 380 1294 435 1766 532 1630 1028 398 841 3493 1181 1771 525 675 377 746 1320 1067 363 282 1629 1647 815 741 8345 510 3923 5284 398 6337  ID  G/L  Chr  Start (bp)  End (bp)  P5 P5 P5 P5 P5 P5 P5 P5 P5 P5 P5 P5 P5 P5 P5 P5 P5 P5 P5 P5 P5 P5 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26  G G L L L L L L L L L L L L L L L L L L L L G G G G G G G G G G G G G G G G G G G G G G G G G  15 15 1 2 3 4 7 7 7 9 10 11 11 12 12 12 14 15 15 17 17 20 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2  30237319 58631770 9725864 203627906 27391786 140187025 72652466 84569635 134815645 37692836 69883760 97278670 110813193 27658181 115508956 117025244 49100288 56648904 91086545 2442274 38288035 29297109 16646685 142533622 145511311 149858970 152160475 154247819 158010337 159680000 161016469 163384213 169651394 173228930 174639051 177764373 179462543 181292771 182037344 187861212 195889657 199215670 1438693 3903894 10828736 13120558 20739093  30597338 59207810 9983505 204011558 27658854 140521672 73120941 84758710 135104307 37940151 70127694 97609941 111560892 27873830 115883865 117328738 49484060 57266624 91292369 2767054 38502716 29933148 17086056 144162829 145872489 151131174 153230241 157619715 158487941 160677252 161777223 167381584 171187941 174094669 177140954 178479748 181045400 181831357 182204185 188252302 196341623 208789026 3489907 9407162 11755093 17177195 23881796  Size (kb) 360 576 258 384 267 335 468 189 289 247 244 331 748 216 375 303 384 618 206 325 215 636 439 1629 361 1272 1070 3372 478 997 761 3997 1537 866 2502 715 1583 539 167 391 452 9573 2051 5503 926 4057 3143  79  ID E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2  G/L  Chr  Start (bp)  End (bp)  G G G G G G G G G G G G G G G G G L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L  19 19 20 20 20 20 20 20 20 21 21 21 22 22 22 22 22 1 1 1 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 4  16184022 52983968 815899 3546156 29486920 43980265 45499042 54011886 58741174 20794703 44701920 45569817 16245214 17053965 28360809 35408079 41150627 5415955 195691334 196414499 55836372 96783694 109113179 158728287 176134627 210844152 5346982 18886339 23277687 26243462 28659721 37346489 68278236 69383522 78394977 83375589 88478268 95948765 100412584 103643599 105677908 110593448 113133331 132236080 166061278 188927793 7507478  18500927 55753488 2441070 4125081 29933148 44352061 47958110 57461196 62435964 21052440 45428172 46927776 16911630 18549230 30166633 41130527 49534385 5868138 196060227 197270713 56202049 96834810 109402865 159062951 176383225 211656085 5765115 19184259 23709450 27403349 30479678 37800287 68785539 71190722 79284456 83859392 90318890 96367298 100932125 104222284 105966136 111713202 113548487 132563562 169039899 189175790 7829767  Size (kb) 2317 2770 1625 579 446 372 2459 3449 3695 258 726 1358 666 1495 1806 5722 8384 452 369 856 366 51 290 335 249 812 418 298 432 1160 1820 454 507 1807 889 484 1841 419 520 579 288 1120 415 327 2979 248 322  ID  G/L  Chr  Start (bp)  End (bp)  E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26  G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G L L L L L L L L L L L  2 2 2 2 2 2 2 2 2 2 2 5 5 5 5 6 6 7 7 7 7 8 8 8 10 10 12 15 15 15 15 15 16 17 21 21 2 2 2 3 3 3 3 4 6 6 6  29024702 36371522 39691420 44824074 47479400 50448852 54385409 56202060 64170018 73670503 217405254 17284044 61430081 85532315 86471625 26747302 158979778 57163665 83422963 125777779 141622813 58157391 133939247 145725741 28063255 48361074 114113748 18272238 20588181 21973970 28106102 30006525 33280312 623188 20794703 28557641 1042562 12063690 18946602 78394977 114814923 170074359 198952699 8979402 13223270 76244437 160616018  31254640 37433458 42547144 46285231 48415259 51101232 55105184 60777537 68144882 75142707 218556881 17553934 61656696 85754045 86737136 27094077 159379225 57368235 83649776 126485305 141981029 58393591 135765484 146208093 28308281 49216977 114564982 19353919 20967788 22386839 28734907 30597338 33741546 962070 21052440 28948912 1404470 12541039 20796325 78583892 115117759 170242091 199276959 9291412 13577224 76653060 161039688  Size (kb) 2230 1062 2856 1461 936 652 720 4575 3975 1472 1152 270 227 222 266 347 399 205 227 708 358 236 1826 482 245 856 451 1082 380 413 629 591 461 339 258 391 362 477 1850 189 303 168 324 312 354 409 424  80  ID E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2  G/L  Chr  Start (bp)  End (bp)  L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L  4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 6 6 6 7 7 8 8 8 8 8 8 8 8 8 8 9 9 9 9 9 9 9 9 10 10 11  13328765 18407291 22415257 63527542 65613252 68980297 78267102 96537113 98579930 107706636 111775984 120125759 128037982 158607566 50839218 66790934 75006893 92624264 107679112 122816388 152030340 57940311 62000083 149900596 11526253 61061239 47568877 60811916 62881808 64395580 71613225 77791301 92229960 116696615 119798295 143780694 937155 7418505 14270695 66254996 67812022 82142726 90861578 124184461 32785206 37069462 48090650  13703700 19814983 23718996 64522776 68082157 71380753 78501811 97073537 99945045 108057925 113738861 121956556 128508036 159783789 51198402 67144270 75588705 92873101 107863907 123306424 152413822 58849697 63102316 150405281 11842183 62176519 47901818 61320284 63455889 64915161 72050929 78175516 92957538 117061503 120752324 144006276 1901823 9455003 14729902 66661410 68974225 82817108 91085013 124626662 33316028 37447817 51464991  Size (kb) 375 1408 1304 995 2469 2400 235 536 1365 351 1963 1831 470 1176 359 353 582 249 185 490 383 909 1102 505 316 1115 333 508 574 520 438 384 728 365 954 226 965 2036 459 406 1162 674 223 442 531 378 3374  ID  G/L  Chr  Start (bp)  End (bp)  E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 E26 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1  L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L G G G G G G G G G G G G G G G  7 7 8 8 8 8 8 8 11 11 11 12 13 15 16 16 16 16 16 16 16 16 16 17 17 17 17 17 18 19 21 22 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2  1499748 84569635 764689 3329536 5449846 6741630 14316570 17007797 69544401 76815285 97154277 32108495 51777911 19842977 20943307 28918888 50079561 62466723 64289064 75492204 78991561 79508959 85109626 810744 4272135 7288226 16857007 32864722 25034674 32504109 33637551 40935061 116637869 155943888 162887424 170814706 173228930 174639051 179462543 200319051 202255347 206141166 232727671 14810177 36371522 46355058 71611728  1737551 84758710 2926879 4564649 6551195 11933605 15321874 18508401 70008693 77212623 97609941 32334573 51987426 20071155 21212616 29239140 56200067 63724558 73888135 78760401 79358404 84688207 88573088 2937807 5025418 14613783 17246408 33700089 25284594 32613567 33992274 41198248 116921215 156178456 163728359 171187941 174094669 175287621 179933730 200612614 203411834 208088220 233105801 18795403 36670189 46727455 72107211  Size (kb) 238 189 2162 1235 1101 5192 1005 1501 464 397 456 226 210 228 269 320 6121 1258 9599 3268 367 5179 3463 2127 753 7326 389 835 250 109 355 263 283 235 841 373 866 649 471 294 1156 1947 378 3985 299 372 495  81  ID E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E2 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28  G/L  Chr  Start (bp)  End (bp)  L L L L L L L L L L L L L L L L L L L L L L L L L L L G G G G G G G G G G G G G G G G G G G G  11 11 11 11 12 12 12 12 14 14 14 15 16 16 16 16 16 17 17 17 18 18 18 19 19 20 20 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 3 3 3 3  54712078 57506078 62532845 96953808 33000844 61666609 85650592 89788969 27311281 38967497 47542813 79676203 29181450 34133153 45866013 49649051 52550335 21762537 22287134 73158376 18245092 23662330 64918775 23775088 50398622 12243506 21691719 5872056 9725864 52141199 63512406 81845355 91947505 148114990 188722502 204625506 7542268 24892640 27134515 47752592 55550156 84979184 203627906 48536891 103294167 128806897 151447608  55479236 58635415 62994414 97518886 34082900 62334361 86993917 90323411 28580622 40587410 48244496 79953718 29392366 34490212 46748378 50083079 52874597 22154806 22595733 73381889 18758389 24490809 65354592 24178465 50652469 12915543 22141882 6741408 9983505 53007168 63808021 82146337 92353749 149276757 189134568 204975414 8181289 26124952 27528584 48098425 55858798 85274881 204180576 49782857 103630934 128962916 151897698  Size (kb) 767 1129 462 565 1082 668 1343 534 1269 1620 702 278 211 357 882 434 324 392 309 224 513 828 436 403 254 672 450 869 258 866 296 301 406 1162 412 350 639 1232 394 346 309 296 553 1246 337 156 450  ID  G/L  Chr  Start (bp)  End (bp)  C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1  G G G G G G G G G G G G G G G G G G G G G G G G G G G L L L L L L L L L L L L L L L L L L L L  2 2 2 2 3 5 6 7 7 7 7 7 7 7 8 11 11 11 14 15 15 15 15 15 17 18 18 1 1 1 1 1 1 2 3 4 4 4 4 4 5 7 8 9 10 11 12  141973184 145486871 159150060 207584984 141103332 68951921 158979778 131677611 141571927 148451510 150112723 153229741 154182039 156304214 58157391 60661723 132424829 133659897 42674393 18272238 28106102 30237319 41491024 86274969 623188 60562274 66303540 8634114 10826432 15428321 19297297 24642063 26424669 18946602 114828518 30307070 33836648 35567290 36170117 140187025 80392139 50158388 86620595 96096087 88056805 97278670 110370511  142221989 145902063 159359345 208085679 141588859 69239886 159379225 132059225 143131253 149799209 151323222 153591526 155735685 158818116 58393591 60880212 133314615 134433506 42924774 19353919 28734907 30597338 41851765 86865897 962070 65581946 67415521 9057702 14457649 16167785 20082385 24852567 27049576 19624269 115117759 33114191 35289377 35995977 37236400 140521672 80686277 50617602 87055819 96322935 88348196 97609941 110713626  Size (kb) 249 415 209 501 486 288 399 382 1559 1348 1210 362 1554 2514 236 218 890 774 250 1082 629 360 361 591 339 5020 1112 424 3631 739 785 211 625 678 289 2807 1453 429 1066 335 294 459 435 227 291 331 343  82  ID E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28  G/L  Chr  Start (bp)  End (bp)  G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G  3 3 4 4 4 4 5 5 5 5 5 6 6 6 6 7 7 7 7 8 8 8 9 9 9 9 10 10 11 11 11 11 11 11 11 12 12 12 12 12 13 14 14 14 15 15 15  155644236 186383958 91190679 124735415 140187025 170275508 57340044 78205814 81026543 85900497 180284904 73242 39928065 45632263 147305808 44733999 104656015 156780334 157782375 21708071 58157391 144602260 36088595 124565721 126474147 138899424 5764881 104813634 50991 3573460 10327230 47194406 57344375 60661723 118808228 52090606 56288136 66689909 108154779 118937352 59352443 19149713 34144592 83790255 21973970 28106102 28918531  156012945 186768122 91570695 125231515 140521672 170915624 57607852 78691735 81370252 86206359 180610179 39555660 45175251 57181727 147798957 45119616 105001474 157071420 158185681 23077984 58393591 145876186 36539166 125173997 126805467 139826635 6282946 105172292 1776138 3835784 10641310 47550302 57551130 61724402 119073431 52382298 56704759 67063558 108581445 121720520 59638075 19485285 34709087 84134919 22386839 28734907 29261049  Size (kb) 369 384 380 496 335 640 268 486 344 306 325 39482 5247 11549 493 386 345 291 403 1370 236 1274 451 608 331 927 518 359 1725 262 314 356 207 1063 265 292 417 374 427 2783 286 336 564 345 413 629 343  ID  G/L  Chr  Start (bp)  End (bp)  C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522  L L L L L L L L L L L L G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G  14 15 16 16 17 17 17 17 17 17 17 19 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 3 3 3 3 3 4 4 4 4 4 4 5 5 5  49100288 36223897 20943307 33659117 810744 3216580 4687532 5418209 9276838 14415762 17035866 55554013 16646685 96864050 142533622 154247819 158010337 159680000 161016469 168402600 173228930 174639051 177945800 182037344 196594358 228205983 233834184 243223806 245724640 8123122 14051860 66790913 102712967 43597989 49579895 52725023 129385338 141103332 1110971 8073005 8617326 9282726 49189866 138441550 12704139 17284044 86471625  49484060 36476298 21212616 34016785 2767054 4200549 5025418 9192837 13532268 16804346 20236821 55707294 17086056 97110652 153692169 157619715 158996174 160677252 167381584 171187941 174094669 176344936 181571274 185060852 227844070 233440847 242043970 244296856 246944046 8616192 18347973 67153990 103110650 43934882 49782857 52967443 129987663 141588859 1847477 8427133 8780507 9841887 49416673 138878456 13007203 17553934 86737136  Size (kb) 384 252 269 358 1956 984 338 3775 4255 2389 3201 153 439 247 11159 3372 986 997 6365 2785 866 1706 3625 3024 31250 5235 8210 1073 1219 493 4296 363 398 337 203 242 602 486 737 354 163 559 227 437 303 270 266  83  ID E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28  G/L  Chr  Start (bp)  End (bp)  G G G G G G G G G G G G G G G G G G L L L L L L L L L L L L L L L L L L L L L L L L L L L L L  15 16 16 16 16 17 17 17 19 19 19 19 19 20 20 20 20 22 1 1 1 2 2 2 2 2 2 2 3 3 3 3 3 3 4 4 4 5 5 5 5 5 6 6 6 7 7  88088780 44231 2425578 29195478 79375882 24695117 69307909 76498848 1331124 9105583 40402918 53658159 57249575 3729010 26104051 34785437 48465589 43225305 4220468 5504245 83905739 79166395 83394849 91608685 94689898 103819868 159197634 211144204 78394977 96819091 114906026 140772863 155328424 163846727 8979402 141770131 157491341 63219983 80232861 89942881 170231354 174498664 149876461 169542240 170655612 8197110 57627188  88498667 1901486 3680499 29881418 79829612 25127880 69931286 78036762 6359433 11860892 41316776 54819578 57517224 4125081 26233197 35853341 49157995 43569976 4552378 5868138 84192189 79443127 83668734 91680249 95017226 104173214 159555327 211423562 78583892 97228441 115117759 141053191 155471419 164196155 9463330 142199996 158162936 63557236 80567551 90441374 170505998 174812270 150732866 169662983 170880179 8445226 58017280  Size (kb) 410 1857 1255 686 454 433 623 1538 5028 2755 914 1161 268 396 129 1068 692 345 332 364 286 277 274 72 327 353 358 279 189 409 212 280 143 349 484 430 672 337 335 498 275 314 856 121 225 248 390  ID  G/L  Chr  Start (bp)  End (bp)  RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522  G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G L L L  5 5 5 6 6 6 6 7 7 7 7 7 7 7 7 8 8 8 11 11 11 11 11 12 12 12 12 13 14 14 14 14 14 15 15 15 16 16 17 17 17 19 20 22 1 2 2  134145577 168095482 170936139 73242 11402225 39096919 158979778 83422963 125777779 128232689 132692815 140395448 147385243 152768219 156529437 36943 6515253 58157391 60661723 78123405 94101838 99935229 108149766 12941004 51140849 107204119 130310595 58396143 19149713 42674393 85052158 93699417 96434336 18473375 41491024 71764685 20137044 33280312 623188 56678248 74090777 47284211 61041281 14410042 25351676 91608685 109113179  134619053 168363001 171218064 3890080 11936033 39555660 159379225 83649776 127812452 132521810 138997094 145320187 151584725 156313401 158818116 179853 6907559 58393591 60880212 93639762 97448007 106974429 124646260 13750814 51672891 107495724 130519529 58861408 19360529 42924774 85476972 95801714 99232080 20338507 41851765 72164226 20628545 33741546 962070 57089085 74851642 47405380 61325197 14647683 25650204 91680249 110005502  Size (kb) 473 268 282 3817 534 459 399 227 2035 4289 6304 4925 4199 3545 2289 143 392 236 218 15516 3346 7039 16496 810 532 292 209 465 211 250 425 2102 2798 1865 361 400 492 461 339 411 761 121 284 238 299 72 892  84  ID E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E28 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20  G/L  Chr  Start (bp)  End (bp)  L L L L L L L L L L L L L L L L L L L L L L G G G G G G G G G G G G G G G G G G G G G G G G G  7 7 8 8 9 10 10 10 10 10 11 11 13 14 14 15 15 18 19 20 20 21 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 4 4 5 5  88304685 158536720 86699540 144157451 67803916 38650109 41947396 50519291 77487773 88945287 39773284 50230022 71226333 32846307 86151504 18272238 91211800 12806854 63079488 10980066 26009233 22979811 5795529 78738027 143665914 204625506 41662 1438693 2444142 4831746 15716583 24892640 36371522 41880246 42852543 44824074 50085577 54239177 60376763 64300715 66790913 49937934 155644236 7937374 54822121 19701106 50423152  88632733 158818116 87055819 144660691 68460734 39116217 42224061 50869761 78116466 89583299 40041456 51421925 71704903 33407161 86629578 18543505 91567261 13066991 63476569 11362367 26227775 23404186 6741408 79075345 143923232 204975414 1218689 2178036 4615940 13719545 23881796 31254640 39527609 42508730 44472090 48731932 51219186 55438372 62598530 66156086 71135781 50482968 156012945 8780507 55235676 20176670 50897952  Size (kb) 328 281 356 503 657 466 277 350 629 638 268 1192 479 561 478 271 355 260 397 382 219 424 946 337 257 350 1177 739 2172 8888 8165 6362 3156 628 1620 3908 1134 1199 2222 1855 4345 545 369 843 414 476 475  ID  G/L  Chr  Start (bp)  End (bp)  RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 RB522 T42 T42 T42 T42 T42 T42 T42 T42 T42 T42 T42 T42 T42 T42 T42 T42 T42 T42 T42 T42 T42 T42 T42 T42 T42 T42 T42 T42 T42 T42 T42  L L L L L L L L L L L L L L L L G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G  3 4 5 6 8 10 11 11 12 12 17 17 17 17 18 19 1 2 2 2 2 2 2 2 2 2 2 2 3 4 5 5 6 6 9 11 11 13 14 14 14 14 14 15 17 18 19  36111427 140187025 68311858 77071746 86620595 64553287 476030 24020342 106476666 117025244 0 1542071 4687532 5418209 16659756 55554013 27020386 7267176 14468368 22667637 30874076 36371522 44563993 97026359 132821463 140471942 201110343 222493290 108016085 8655779 68951921 180195851 62000083 158818189 10178412 60661723 131887972 63561919 49448838 58300168 63455662 71880540 88433473 30006525 623188 42657928 8629145  36427114 140521672 69102023 77347942 87055819 64995092 23649364 32382584 106770408 117328738 695461 4200549 5025418 18110985 17277836 55707294 27234980 9407162 17494103 23428639 31142648 36799901 45938104 98604893 134996036 140728943 201426850 222928085 108230341 9129871 70342024 180444136 62065546 159379225 10618299 60880212 132151114 64068098 57610292 62987077 71665768 87964669 106318374 30597338 962070 42840984 8832847  Size (kb) 316 335 790 276 435 442 23173 8362 294 303 695 2658 338 12693 618 153 215 2140 3026 761 269 428 1374 1579 2175 257 317 435 214 474 1390 248 65 561 440 218 263 506 8161 4687 8210 16084 17885 591 339 183 204  85  ID E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20  G/L  Chr  Start (bp)  End (bp)  G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G  5 5 6 6 6 6 6 6 6 7 8 8 9 9 9 10 11 11 11 12 12 12 12 12 13 13 14 15 15 15 15 15 15 16 16 17 17 17 17 18 19 19 19 19 19 20 20  85900497 95697117 73242 21122117 25059720 29457137 39928065 45632263 56250565 48717014 49137279 54928152 36088595 96551213 138368718 24794390 2055967 60661723 79872816 19062093 31699805 52090606 115064470 129125980 58515152 63561919 64383439 20302216 21973970 30006525 62785585 75603709 83040659 4441455 88443716 623188 7205406 16624310 74307357 25034674 2928774 5172606 10040796 18121106 18873669 4484372 31716051  86206359 95852946 20763610 24801727 29123783 39555660 44815780 53956516 57181727 49076562 49417714 55191935 36774290 97039243 138919624 25038334 3183753 60880212 80194218 19403874 31917245 52327387 115347630 129776292 58861408 64068098 64555988 20627698 22386839 30597338 63049771 75885169 83217528 4947778 88573088 1865526 8079234 17035446 74959077 25284594 3354214 8832847 11860892 18643264 19630711 5049159 32417420  Size (kb) 306 156 20690 3680 4064 10099 4888 8324 931 360 280 264 686 488 551 244 1128 218 321 342 217 237 283 650 346 506 173 325 413 591 264 281 177 506 129 1242 874 411 652 250 425 3660 1820 522 757 565 701  ID  G/L  Chr  Start (bp)  End (bp)  T42 T42 T42 T42 T42 T42 T42 T42 T42 T42 T42 T42 T42 T42 T42 T42 T42 T42 T42 T42 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5  G G L L L L L L L L L L L L L L L L L L G G G G G G G G G G G G G G G G G G G G G G G G G G G  20 21 1 2 2 2 2 2 2 2 3 6 7 8 8 10 12 15 15 19 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2  33618231 19095584 25351676 11750125 14137447 17417308 18946602 229910358 237043334 239997825 140772863 13223270 150155467 58157391 86724325 4260820 47207929 19842977 21945545 32504109 16646685 142533622 145511311 150310886 158758725 174639051 179462543 187522092 189269177 190496532 196195485 215473404 216564705 221391322 243722287 246181048 5090758 15467410 21054372 51035350 78140908 87512069 103493167 144430193 145486871 191147109 222240742  33841512 19368540 25650204 13079174 14464191 18347973 21011770 230310424 239819601 241063385 141053191 13577224 150543808 58393591 87055819 4547435 47466311 20071155 22386839 32613567 17086056 145133682 147083700 158487941 163430309 176344936 187248945 188584062 189714992 194595123 214702922 215816541 220169719 242043970 244912908 246944046 5891801 16651868 21296906 51768164 78929009 87846427 104173214 144816425 145902063 191549049 222928085  Size (kb) 223 273 299 1329 327 931 2065 400 2776 1066 280 354 388 236 331 287 258 228 441 109 439 2600 1572 8177 4672 1706 7786 1062 446 4099 18507 343 3605 20653 1191 763 801 1184 243 733 788 334 680 386 415 402 687  86  ID E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 E20 P4 P4 P4 P4  G/L  Chr  Start (bp)  End (bp)  G L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L G G G G  22 1 1 2 2 3 3 3 5 5 5 5 6 6 6 6 7 7 7 7 7 7 8 8 8 8 10 10 10 10 11 11 11 11 11 12 12 13 16 18 18 19 20 1 1 1 1  43225305 12724432 104202532 42339545 211144204 44892353 78394977 114906026 12026212 20112325 30331484 98983920 57571522 103057262 149900596 160616018 1499748 18699485 50158388 57627188 61061239 158298447 6741630 43185803 85434907 91756147 38650109 50596911 54832926 77487773 3414002 10665713 34772104 50230022 125166505 32108495 46772772 41211420 33659117 12806854 15026396 55554013 939429 16646685 116927827 143665914 232830875  43569976 13569886 104657658 42688302 211423562 45416063 78583892 115117759 12326896 20653881 30610845 99378879 57829665 103422050 150732866 161039688 1737551 19259911 50617602 58017280 62176519 158687174 8080568 43912178 87055819 92017226 39116217 50869761 55256917 77592293 3661668 10755802 35267094 51302042 125470718 32334573 47154491 41424564 33927835 13135104 15320434 55707294 2441070 17086056 117385135 143791318 233105801  Size (kb) 345 845 455 349 279 524 189 212 301 542 279 395 258 365 832 424 238 560 459 390 1115 389 1339 726 1621 261 466 273 424 105 248 90 495 1072 304 226 382 213 269 328 294 153 1502 439 457 125 275  ID  G/L  Chr  Start (bp)  End (bp)  T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5  G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G  3 3 3 3 3 3 3 3 4 4 4 4 4 4 5 5 5 5 5 6 6 6 6 6 6 6 7 7 7 7 8 8 8 8 8 9 9 9 10 10 11 11 12 12 12 12 12  82058696 85873782 109747007 114906026 130702173 165585035 188200702 190572724 8073005 13472033 48771496 117123171 126043717 151664080 17284044 51281449 86471625 97652683 180195851 48501263 49184078 54592590 65932130 79052729 81288115 152028464 15542301 61087267 86477382 114491027 18242281 28931963 83316594 106904387 113870788 9588884 24798764 72806252 15913133 61498616 100189595 100820386 17318075 36302222 73200372 77766046 83504953  83273329 86189057 109971516 115117759 131070392 165967707 188562337 190902546 8427133 13770492 49210770 117838327 126776025 152203871 17553934 51628988 86737136 97856979 180444136 48963512 49461459 54729884 66468315 79753932 81664223 152461187 15793197 61270796 86755032 115586861 18508401 29222390 83767100 107311076 114193060 10246475 25832442 73010767 16440716 62052694 100588796 101077028 18090825 36432239 73777803 78269342 83977622  Size (kb) 1215 315 225 212 368 383 362 330 354 298 439 715 732 540 270 348 266 204 248 462 277 137 536 701 376 433 251 184 278 1096 266 290 451 407 322 658 1034 205 528 554 399 257 773 130 577 503 473  87  ID P4 P4 P4 P4 P4 P4 P4 P4 P4 P4 P4 P4 P4 P4 P4 P4 P4 P4 P4 P4 P4 P4 E24 E24 E24 E24 E24 E24 E24 E24 E24 E24 E24 E24 E24 E24 E24 E24 E24 E24 E24 E24 E24 E24 E24 E24 E24  G/L  Chr  Start (bp)  End (bp)  G G G G G G G G G G G G G G L L L L L L L L G G G G G G G G G G G G G G G G G G G G G G G G G  3 4 5 5 5 5 6 14 15 15 15 16 20 22 3 3 4 6 12 15 18 22 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 4 5 5  75285656 8617326 17284044 49960569 71207958 134642615 73242 19149713 21973970 28106102 30006525 33280312 53300231 17053965 78394977 114828518 8979402 13223270 32108495 19884809 12806854 40935061 16638721 120249993 142533622 144594746 145425752 156649526 158010337 160338731 161558031 162887424 173658230 174639051 179462543 199215670 202255347 204118116 206141166 212028920 214753241 232727671 243881127 121240000 108599229 16358405 17284044  75515882 8780507 17553934 50290883 71428448 134881601 346084 19485285 22386839 28734907 30597338 33741546 54270330 17206783 78583892 115117759 9291412 13524298 32334573 20071155 13135104 41198248 17086056 120932201 144162829 144873033 145872489 157113477 158283641 160677252 161777223 163728359 174094669 176344936 179933730 199653218 203411834 205439437 207970850 212449749 215373858 233105801 244296856 121633928 108797097 16598168 17553934  Size (kb) 230 163 270 330 220 239 273 336 413 629 591 461 970 153 189 289 312 301 226 186 328 263 447 682 1629 278 447 464 273 339 219 841 436 1706 471 438 1156 1321 1830 421 621 378 416 394 198 240 270  ID  G/L  Chr  Start (bp)  End (bp)  T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T5  G G G G G G G G G G G G G G G G G G G G G G G G G G G G L L L L L L L L L L L L L L L L L L L  12 12 12 12 13 13 13 13 14 14 14 14 14 14 16 16 16 17 18 18 18 18 18 19 21 21 21 22 1 1 1 2 2 3 3 3 3 5 5 7 8 8 8 10 10 11 12  86487712 89886535 90278355 128150230 59084555 64317369 67175227 82307672 26401540 41518987 42674393 70137529 80658075 99356742 21939143 52309608 64588814 48684140 5362621 36430587 43069061 61022046 69281624 7749446 19365844 44616528 45569817 36007572 30216330 65050275 178395125 17341117 51706280 44892353 73471190 152832716 190306876 37052109 103018513 24242808 7035076 75167545 89314331 124792962 127834059 129706418 79917287  86754944 90205587 90618336 128548133 59396464 64544139 67681892 82609458 27384263 42281787 42924774 70215232 80879562 99584727 22187488 52599483 65053676 49205759 5746803 36743158 43436781 61554258 69613151 7984147 19698905 45041136 45747046 36324767 30537929 65530390 178621310 17715047 52051295 45303025 73915690 153205949 190600506 37473658 103426734 24445001 7289755 75553560 89669378 126788208 128349027 130119560 80564119  Size (kb) 267 319 340 398 312 227 507 302 983 763 250 78 221 228 248 290 465 522 384 313 368 532 332 235 333 425 177 317 322 480 226 374 345 411 445 373 294 422 408 202 255 386 355 1995 515 413 647  88  ID E24 E24 E24 E24 E24 E24 E24 E24 E24 E24 E24 E24 E24 E24 E24 E24 E24 E24 E24 E24 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6  G/L  Chr  Start (bp)  End (bp)  G G G G G G G G G G L L L L L L L L L L G G G G G G G G G G G G G G G G G G G G G G G G G G G  8 13 13 13 13 13 14 14 14 18 4 10 11 12 13 16 22 22 22 22 1 1 1 1 1 1 1 2 2 2 2 2 3 5 5 6 7 7 7 7 7 7 8 9 9 9 9  144158852 100030217 102377421 106484325 108629457 113492713 19149713 21763281 105584423 42657928 140187025 48361074 97278670 92113258 62267126 20943307 31240461 32217850 36928815 41907119 1058909 8127005 26424669 39063444 44687143 232727671 245436183 9985433 24892640 84979184 208050137 232967040 121682380 1019971 175623077 41352064 6038742 57194816 71946110 72809108 100027063 129173245 144602260 33783560 35583889 129488250 133976381  144660691 101730172 102768830 107155648 112845578 114093192 19485285 22006855 105967733 42803873 140521672 49013085 97609941 92516893 62577460 21212616 32005256 36515461 37572224 49534385 3000997 9983505 29453636 40504667 45325761 233546093 245747506 11755093 25572843 85274881 208489071 233991801 121964439 1562826 176701325 43237228 6960042 57562822 72215337 73534009 102036700 129934305 146208093 34331118 36539166 132999756 136359804  Size (kb) 502 1700 391 671 4216 600 336 244 383 146 335 652 331 404 310 269 765 4298 643 7627 1942 1857 3029 1441 639 818 311 1770 680 296 439 1025 282 543 1078 1885 921 368 269 725 2010 761 1606 548 955 3512 2383  ID  G/L  Chr  Start (bp)  End (bp)  T5 T5 T5 T5 T5 T5 T5 T5 T5 T5 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25  L L L L L L L L L L G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G L L  15 15 16 16 16 17 18 19 20 20 1 2 2 3 3 4 5 5 5 5 6 7 8 8 10 10 10 11 11 12 12 12 15 15 15 16 16 17 17 18 18 19 19 19 21 1 2  57743715 73569812 58148238 71288486 84535576 2534851 22079376 45512392 42615749 55362290 50760069 14410329 23828557 78049335 190572724 65966121 17284044 97652683 105950837 180195851 54803239 61061239 18242281 96606103 16290075 28063255 118147658 22289940 131887972 32399702 36302222 130568951 18473375 19087319 30237319 20943307 64588814 623188 55638613 20449482 42657928 8934066 23835271 55554013 20794703 87202895 46355058  58409873 74091082 58608110 72135811 85564330 2937807 22624097 45693347 42799481 55807791 51272846 16914958 24216814 78310037 190793756 66436409 17553934 97856979 106368860 180444136 55088907 62784594 18508401 96811332 16607824 28308281 118360397 22485886 132151114 32687563 36432239 130978591 18717253 19353919 30597338 21212616 64837478 962070 56233848 20735586 43072296 9026570 24178465 55707294 21052440 87588045 46727455  Size (kb) 666 521 460 847 1029 403 545 181 184 446 513 2505 388 261 221 470 270 204 418 248 286 1723 266 205 318 245 213 196 263 288 130 410 244 267 360 269 249 339 595 286 414 93 343 153 258 385 372  89  ID E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6  G/L  Chr  Start (bp)  End (bp)  G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G L  9 11 11 11 11 11 11 11 12 14 15 15 15 16 16 16 16 16 17 17 17 17 17 17 17 17 17 17 18 18 18 18 19 19 19 19 19 19 20 20 21 21 21 22 22 22 1  138368718 50991 47194406 61823795 62944653 69117677 72553832 118055345 119183787 22869703 30328715 39080446 72775397 44231 11419973 29306982 30539947 87260490 0 1604505 3295734 16857007 26042085 43090007 45153711 54447833 69977768 73871046 3167304 11684403 16897445 25034674 74843 1331124 10458166 16184022 50652470 53243799 54443015 61386952 20794703 43899532 46246247 17475778 35061869 43225305 100794896  139959845 1875933 47629247 62236904 67116241 69880011 73412975 118758082 122155980 23236087 30597338 39391363 73217136 3680499 12014547 29880154 31499638 88573088 1628597 2767054 4904795 17246408 26307306 45085833 46634199 54973480 73230854 78615238 3472372 12100695 18303681 25284594 1095508 6694652 11737906 18643264 52308679 55203857 54747849 62435964 21052440 44194069 46694679 18674825 41802041 46032632 101075341  Size (kb) 1591 1825 435 413 4172 762 859 703 2972 366 269 311 442 3636 595 573 960 1313 1629 1163 1609 389 265 1996 1480 526 3253 4744 305 416 1406 250 1021 5364 1280 2459 1656 1960 305 1049 258 295 448 1199 6740 2807 280  ID  G/L  Chr  Start (bp)  End (bp)  T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T25 T14 T14 T14 T14 T14 T14 T14  L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L G G G G G G G  2 2 2 2 3 3 3 3 4 5 5 6 6 7 7 8 9 9 10 10 10 10 10 10 11 11 11 11 12 12 12 13 16 16 17 18 18 18 20 22 1 2 2 3 4 4 4  87057872 120934194 134784717 217757668 38457066 78394977 129385338 140772863 3362479 15782020 134308327 94071515 169888230 149177480 158404469 764689 82833412 95274007 77123718 79607481 110633487 124792962 129620434 132635360 6270497 114281321 118980810 132051142 34127668 100290871 112894158 110703658 75550721 83873509 2534851 25034674 36149440 44137937 47317703 17053965 33572793 15716583 214907768 190572724 48771496 171943081 177422553  87638682 121441961 135096043 218352598 38837528 78583892 129741914 141053191 3610639 16253318 135430819 94355791 170479234 150001617 158818116 1142225 83098662 95599862 77578274 81420249 111047441 126455020 131698871 135279687 6580309 114600734 119354299 132915120 34451327 100633658 113314551 112948798 75802088 88336665 2937807 25284594 36407233 44552924 47816133 17206783 33943747 18181769 215352824 190793756 49210770 172140052 177909585  Size (kb) 581 508 311 595 380 189 357 280 248 471 1122 284 591 824 414 378 265 326 455 1813 414 1662 2078 2644 310 319 373 864 324 343 420 2245 251 4463 403 250 258 415 498 153 371 2465 445 221 439 197 487  90  ID E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E6 E21 E21 E21 E21  G/L  Chr  Start (bp)  End (bp)  L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L G G G G  1 1 2 2 2 2 2 3 3 3 3 4 5 5 5 5 5 6 6 6 6 8 8 8 8 9 9 11 11 11 11 12 14 15 16 17 17 18 18 18 19 19 20 1 1 1 1  167301416 171178733 1438693 77054044 97026359 109113179 209283920 26243462 80918434 89945475 110593448 191006683 49479231 75006893 100253676 170231354 180093032 57874178 62000083 122778393 149900596 47568877 77791301 86699540 90405349 66254996 67803916 54712078 62532845 93414925 96953808 69844305 38847479 79676203 33659117 22287134 73158376 15026396 40025831 63741633 23835271 50398622 4968909 145425752 150080115 157848491 160338731  167654916 171743135 1649373 77302660 97280716 109402865 209965965 27041194 81176295 90318890 111097910 191137774 49913062 75588705 100665725 170505998 180343927 58849697 62603705 123049799 150732866 47901818 78175516 87055819 90822242 66661410 70224724 55479236 62994414 93936247 97518886 70114686 39960099 79953718 34016785 22595733 73381889 15320434 40393746 63919160 24178465 50652469 5343849 145872489 156658752 159891049 160958046  Size (kb) 354 564 211 249 254 290 682 798 258 373 504 131 434 582 412 275 251 976 604 271 832 333 384 356 417 406 2421 767 462 521 565 270 1113 278 358 309 224 294 368 178 343 254 375 447 6579 2043 619  ID  G/L  Chr  Start (bp)  End (bp)  T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14  G G G G G G G G G G G G G G G G G G G G G G G G G G G G G L L L L L L L L L L L L L L L L L L  4 4 4 4 5 5 5 5 5 7 7 7 8 8 9 10 12 12 14 15 15 15 15 17 18 19 19 20 20 1 2 2 3 4 4 5 5 7 7 9 11 11 11 11 11 11 11  183340483 185082952 187161436 190853246 17284044 20897033 41237134 69602994 180093032 57163665 64972395 109009814 12999086 92381128 139979344 50596911 85650592 130568951 85765244 18674575 50899677 59960618 86073780 27961446 42657928 47912047 55554013 24766816 40126629 116637869 29678589 223727102 145364190 142631188 172058782 61657012 62959058 7376245 16589721 97212464 50991 2197942 7926129 14183583 16945105 30243665 44571231  184029894 185401436 187685225 191137774 17553934 21399027 41668526 70066836 180444136 57368235 65209849 109465769 13429882 92957538 140185705 50869761 86068176 130978591 86154997 20053874 51145331 60314021 86865897 28214642 43436781 48626174 55707294 25046218 40500600 116921215 30015687 224009643 145595605 142828372 172379103 61943229 63386658 7947720 16636674 97444301 1776138 5839559 13369379 16057348 20006238 35665813 44925353  Size (kb) 689 318 524 285 270 502 431 464 351 205 237 456 431 576 206 273 418 410 390 1379 246 353 792 253 779 714 153 279 374 283 337 283 231 197 320 286 428 571 47 232 1725 3642 5443 1874 3061 5422 354  91  ID E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21  G/L G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G  Chr  Start (bp)  End (bp)  1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 4 4 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6 6 7  166264720 176574921 179640235 181937518 198461529 201646252 209696870 223536766 228205983 229883839 232727671 242294867 242997307 1438693 7542268 9985433 15716583 19690629 23361965 28214990 42999225 45282682 54031551 71611728 94986258 139134656 143938661 150646482 417247 5927785 17284044 55199515 63789206 82802679 95697117 116123966 73242 6362577 14674050 21356155 25059720 33017597 39096919 39928065 45494917 51704583 44818259  167381584 176971757 181831357 182204185 201431856 208088220 212332499 228168309 229467946 231685446 233391573 242642426 244387464 2793742 8825151 11755093 16914958 20796325 23881796 28865200 43518222 46285231 55105184 72007879 95863637 139386613 144222024 151003446 1847477 6293125 17553934 55485623 64142333 83034279 95852946 116386009 5789420 14506540 18121480 21654555 32151955 38660643 39555660 45175251 46734973 53956516 45119616  Size (kb) 1117 397 2191 267 2970 6442 2636 4632 1262 1802 664 348 1390 1355 1283 1770 1198 1106 520 650 519 1003 1074 396 877 252 283 357 1430 365 270 286 353 232 156 262 5716 8144 3447 298 7092 5643 459 5247 1240 2252 301  ID  G/L  Chr  Start (bp)  End (bp)  T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 T14 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3  L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L G G G G G G G G G G G G G G G G  11 11 11 11 11 11 11 11 11 12 12 12 12 12 15 16 16 16 16 16 16 16 16 16 17 17 18 19 19 19 22 1 1 1 2 2 2 3 6 7 7 8 9 9 9 11 11  68457614 72553832 74847555 97154277 102841892 104388385 109535513 114281321 132051142 42172642 59957940 79773979 102225696 117025244 39035666 47457492 50079561 53615738 60515970 68719438 70168706 75370384 75886028 87165550 2534851 35065361 25034674 17227985 20479187 32504109 17053965 52663511 78738027 177764373 7750863 198492712 241714149 49579895 147323170 72809108 151676150 54928152 25318379 35286076 130791422 60661723 131887972  71041286 73412975 78921469 97534210 103303951 106138665 113295360 131598665 132915120 42629204 60195470 80088388 102559169 117328738 39391363 48858380 52510034 59512370 66485262 69159549 73945290 75802088 85564330 87542642 2937807 35304654 25284594 17558373 20771666 32613567 17206783 53007168 79075345 178479748 8181289 198759383 242006836 49782857 147837918 73201519 151985718 55339325 25715548 35798430 131615932 60880212 132389201  Size (kb) 2584 859 4074 380 462 1750 3760 17317 864 457 238 314 333 303 356 1401 2430 5897 5969 440 3777 432 9678 377 403 239 250 330 292 109 153 344 337 715 430 267 293 203 515 392 310 411 397 512 825 218 501  92  ID E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21  G/L  Chr  Start (bp)  End (bp)  G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G L L L  7 7 7 8 8 8 9 11 11 11 12 12 13 14 14 14 15 15 15 15 15 15 15 15 15 16 17 17 17 17 17 17 17 17 17 18 18 19 19 19 20 20 21 22 2 2 2  73680992 124677369 127398355 106601289 120711365 140294522 123326445 1321707 57344375 63321142 32254703 71994675 102618440 19497166 76307165 89811265 18898414 21973970 26275701 28106102 39724582 71764685 73336790 75603709 96709108 52079281 6383241 36547206 39434414 45294025 56558187 65201407 69307909 73871046 75249971 20449482 42657928 1331124 13709724 63435376 3729010 60809452 44152494 37376687 35571545 75857509 78140908  73956925 124943364 127812452 107311076 121163538 141002709 123648944 1660253 57551130 64621654 32687563 72180449 102958785 19718160 76655057 90956790 19133581 22386839 26597300 28691954 40237983 72403143 73793805 75885169 97132862 52360249 7387494 38155435 40139868 45685773 57247862 65563337 73457154 74851642 78615238 21010506 42803873 2196817 15301063 63654245 4125081 61325197 44522210 38610512 35837286 77204381 81001459  Size (kb) 276 266 414 710 452 708 322 339 207 1301 433 186 340 221 348 1146 235 413 322 586 513 638 457 281 424 281 1004 1608 705 392 690 362 4149 981 3365 561 146 866 1591 219 396 516 370 1234 266 1347 2861  ID  G/L  Chr  Start (bp)  End (bp)  P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3  G G G G G G G G G G G G G G G L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L  12 12 13 13 14 15 15 15 15 15 16 17 17 19 19 1 1 2 2 2 2 3 3 3 3 3 4 5 5 5 6 6 6 7 7 8 8 8 11 11 11 11 11 11 12 12 12  1526627 12243842 57053680 59352443 24926668 21945545 28106102 30237319 32897394 91086545 1341140 4303684 35490927 51436748 60306236 104420480 106860717 1042562 59571563 199463802 211144204 30099494 75285656 78394977 107037366 140772863 108039548 49479231 62884026 102114463 67474215 149900596 170655612 1499748 121931947 36186658 43185803 85434907 40510250 46058617 47474531 49344159 50230022 97154277 62057653 106476666 107204119  1719982 12729565 57436388 59638075 25366361 22386839 29121290 30597338 33198054 91292369 1901486 4700666 35997410 51896236 60867589 104657658 107290858 1404470 59886525 199805543 211540445 30364799 75515882 78583892 107517939 141053191 108580318 49636937 63168836 102519831 67751642 150732866 170880179 1737551 122282009 36516657 43912178 85823834 40852607 46407944 47860662 49626739 51192828 97518886 62430133 106770408 107495724  Size (kb) 193 486 383 286 440 441 1015 360 301 206 560 397 506 459 561 237 430 362 315 342 396 265 230 189 481 280 541 158 285 405 277 832 225 238 350 330 726 389 342 349 386 283 963 365 372 294 292  93  ID E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 E21 T3 T3 T3 T3 T3 T3 T3  G/L  Chr  Start (bp)  End (bp)  L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L G G G G G G G  2 2 2 2 3 3 3 4 4 5 5 6 7 7 7 7 8 8 8 8 8 10 10 10 11 11 11 11 11 12 12 12 14 16 18 18 18 19 19 21 1 1 3 5 5 5 5  82120028 145486871 179449929 211144204 78394977 117931539 140772863 132667102 180427322 66790934 95842127 67459297 1499748 11969246 61087267 87945541 36095236 43185803 63189500 86196327 117969492 41947396 66355758 77389763 3414002 18783635 47479617 84053362 97084524 32108495 36302222 103566212 40026915 33659117 16659756 56366046 57739648 8934066 55554013 23153285 5116964 120047377 48134752 68905030 82709486 143388623 180195851  83668734 146254326 179831272 211656085 78583892 118057362 141053191 133645430 181366822 67144270 96089798 67668616 1737551 12200051 62176519 88632733 36516657 43912178 63663884 87055819 118356842 42224061 66994232 77592293 3661668 19003338 47860662 84505710 97518886 32334573 36432239 104021895 40587410 35136830 16944006 56761467 58026453 9026570 55707294 23404186 5290233 120331938 48468265 69102023 82952978 143726339 180444136  Size (kb) 1549 767 381 512 189 126 280 978 940 353 248 209 238 231 1089 687 421 726 474 859 387 277 638 203 248 220 381 452 434 226 130 456 560 1478 284 395 287 93 153 251 173 285 334 197 243 338 248  ID  G/L  Chr  Start (bp)  End (bp)  P3 P3 P3 P3 P3 P3 P3 P3 P3  L L L L L L L L L  13 14 17 17 18 19 19 19 19  64317369 64454982 22287134 65056159 12806854 910519 8934066 32504109 55554013  64544139 64964479 22595733 65563337 13135104 1369051 9026570 32613567 55707294  Size (kb) 227 509 309 507 328 459 93 109 153  94  ID T3 T3 T3 T3 T3 T3 T3 T3 T3 T3 T3 T3 T3 T3 T3 T3 T3 T3 T3 T3 T3 T3 T3 T3 T3 T3 T3 T3  G/L  Chr  Start (bp)  End (bp)  G G G G G G G G G G G G L L L L L L L L L L L L L L L L  7 7 11 12 13 14 14 15 15 15 15 16 2 2 3 3 4 4 5 6 6 7 8 9 10 13 19 19  57163665 64972395 131887972 29727999 63561919 19149713 104967556 18473375 21973970 30237319 36223897 33280312 210209170 211144204 78394977 140772863 8979402 48771496 80392139 13223270 160616018 143515150 86699540 82833412 4158992 51777911 8629145 32504109  57368235 65209849 132151114 29929154 64068098 19485285 105600462 20053874 22386839 30597338 36476298 33584068 210467608 211349074 78583892 141053191 9291412 49210770 80686277 13524298 161039688 143839638 87055819 82953738 4425972 52002865 8784792 32613567  Size (kb) 205 237 263 201 506 336 633 1580 413 360 252 304 258 205 189 280 312 439 294 301 424 324 356 120 267 225 156 109  Table A.2  aCGH alterations by sample (RB1-/-).  ID  G/L  Chr  Start (bp)  End (bp)  Size  ID  G/L  Chr  Start (bp)  End (bp)  Size  T23 T23 T23 T23 T23 T23 T23 T23 T23 T23 T23 T23 T23 T23 T23 T23 T23 T23 T23 T23 T23 T23 T23 T23 T23 T23 T23 T23 T23 T23 T23 T23 T23 T23 T23 T23 T23 T23 T23 T23 T23 T23 T23 T23 T23  G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G L L L L L L L  1 2 2 3 3 5 5 5 5 6 6 6 6 6 6 6 7 11 12 12 12 12 12 12 14 15 16 16 18 18 18 18 18 18 18 18 19 22 1 1 4 8 8 9 9  220760289 6372092 120641847 21893268 141103332 568397 17284044 20897033 49960569 73242 15729721 19638959 27295136 33848331 103346894 170655612 57163665 134225567 71994675 112894158 122604516 125926009 127185807 130113790 19149713 21973970 33400710 50551653 1946123 7039213 10433893 31135911 42007182 45570558 69281624 72745519 36871853 14410042 62245042 152139606 69910765 86196327 134535936 29711074 113299231  221225248 6738494 121052681 22110528 141588859 1013978 17553934 21399027 50670962 346084 16332107 24574853 27628148 35024860 103604028 170880179 57368235 134433506 72180449 116351108 125418667 126964150 128548133 131701464 19485285 22386839 33741546 50792425 2506229 8637511 11111665 35657079 44723913 46140574 72148783 76098439 37049593 14660949 62523933 152376962 70332437 87506471 134863249 30043324 113848887  (kb) 465 366 411 217 486 446 270 502 710 273 602 4936 333 1177 257 225 205 208 186 3457 2814 1038 1362 1588 336 413 341 241 560 1598 678 4521 2717 570 2867 3353 178 251 279 237 422 1310 327 332 550  T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66  G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G L L  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 3 3 4 5 5 6 6 7 11 14 14 14 14 14 14 14 14 14 15 16 17 17 18 22 1 1  27020386 142652783 150229090 155801127 158758725 161016469 165155625 174041164 177864963 179640235 189269177 190304054 196799511 210772211 216039670 222704527 228205983 229174289 242089298 142345971 175445960 143031922 34094987 180195851 73242 36620128 141962686 57344375 53626221 60182950 65111104 73806697 77836558 80600513 86623012 96121656 104967556 30237319 64588814 10094466 33264100 42657928 17053965 617 4924403  27234980 142998578 155733368 158283641 160677252 163072819 169658465 175146843 178479748 183513118 189714992 191386589 209169786 215373858 220609576 227325091 229034632 234815927 245339706 142646405 175738257 143345850 34371608 180444136 36488793 58849697 142316803 57551130 55640667 64555988 71263950 77513223 80558546 86154997 95405991 103750538 106318374 30539910 65053676 10520549 33700089 42803873 17206783 3000997 5088349  (kb) 215 346 550 248 191 205 450 110 615 387 446 108 123 460 457 462 829 564 325 300 292 314 277 248 364 222 354 207 201 437 615 370 272 555 878 762 135 303 465 426 436 146 153 300 164  95  ID  G/L  Chr  Start (bp)  End (bp)  Size  ID  G/L  Chr  Start (bp)  End (bp)  Size  T23 T23 T23 T23 T23 T23 T23 T23 T23 T23 T23 T23 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53  L L L L L L L L L L L L G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G  11 11 12 13 13 13 13 13 13 13 15 19 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3  97278670 125166505 32108495 47570684 59352443 66263513 67175227 70429098 87396186 92154455 19884809 32504109 36466785 46604573 142533622 155029936 161016469 168471887 173228930 174639051 177945800 179462543 196195485 199539859 202916221 222704527 224564745 226511813 240316624 245724640 41662 5541877 29403359 36442569 46355058 71611728 73898940 91021063 113143567 120934194 128716306 179059362 218193818 236568755 46140 2804349 13499970  97534210 125470718 32334573 47823050 59961173 66805219 67681892 73462825 87732535 92774084 20071155 32613567 37675114 48257830 153559241 160677252 167445692 171525137 174053395 176344936 178479748 184426068 198714418 201882048 220609576 223152230 225431615 233440847 242491480 246944046 317294 6228194 29872762 36996317 46727455 72279682 74604122 91590359 114143148 121633928 129315130 179486309 218637368 238194747 2390404 6226254 14924624  (kb) 256 304 226 252 609 542 507 3034 336 620 186 109 1208 1653 11026 5647 6429 3053 824 1706 534 4964 2519 2342 17693 448 867 6929 2175 1219 276 686 469 554 372 668 705 569 1000 700 599 427 444 1626 2344 3422 1425  T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T66 T4 T4 T4 T4 T4 T4 T4 T4 T4 T4 T4  L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L G G G G G G G G G G G  1 1 1 2 2 3 4 4 5 5 5 8 12 13 13 13 13 13 13 13 13 16 16 16 16 16 16 16 16 16 16 16 16 17 17 18 1 1 1 1 1 1 1 1 1 1 1  17486898 29953410 104001576 88921446 223605559 150185459 5574550 49189866 80319034 137083206 143610226 86620595 120601206 36678499 41211420 43481375 46168557 48654459 54559086 57053680 58515152 10586248 12340398 26891980 53615738 63381801 64289064 64890123 71288486 76619437 77371695 79053536 79901920 0 9663047 23570175 617 2341648 3204885 12343280 27020386 29573058 74438002 101933924 106221897 154928108 155661462  19020943 30239890 104300583 89405301 224009643 150424655 5959685 49416673 80686277 137340623 143913187 87055819 120855003 40124114 43127842 45768530 46488557 52002865 54932279 57436388 61033077 11089099 12607349 29392366 54216885 63762681 64554473 65208073 72135811 77228585 77830699 79572749 85564330 5025418 9930706 23897445 1487588 3000997 3514176 12747440 27330241 30009232 74628468 102562274 106419881 155216620 155849211  (kb) 153 286 299 484 404 239 385 227 367 257 303 435 254 344 191 228 320 334 373 383 251 503 267 250 601 381 265 318 847 609 459 519 566 502 268 327 148 659 309 404 310 436 190 628 198 289 188  96  ID  G/L  Chr  Start (bp)  End (bp)  Size  ID  G/L  Chr  Start (bp)  End (bp)  Size  T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53  G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G  3 3 3 3 3 3 3 3 4 4 4 4 5 5 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 7 7 7 7 7 8 8 8  72602189 75285656 108872278 114906026 124106796 126980939 129122714 141082476 3362479 9051500 119557416 188225705 1684158 7229957 17284044 67361489 68951921 85900497 132432815 134308327 139059617 163102487 175953307 180195851 73242 14319218 15450735 22866262 25059720 29457137 35048432 43065666 45632263 51704583 53635140 55796805 58230371 64983144 167951793 1803168 76182168 126130514 131677611 157061042 7035076 9929219 36772374  72944366 75515882 109483457 115117759 125664529 128752028 131723522 141588859 4065877 9841887 119899496 188555694 2928706 7549647 17553934 67899255 70342024 86206359 133318206 137208902 139415354 163429773 176299886 180715161 13658686 14506540 20622576 24238752 27094077 34703255 42527171 44815780 49681191 52998900 54076882 56001013 58849697 65308099 170880179 2241382 76393428 126748077 132059225 158818116 8212432 11424992 37157205  (kb) 342 230 611 212 1558 1771 2601 506 703 790 342 330 1245 320 270 538 1390 306 885 2901 356 327 347 519 13585 187 5172 1372 2034 5246 7479 1750 4049 1294 442 204 619 325 2928 438 211 618 382 1757 1177 1496 385  T4 T4 T4 T4 T4 T4 T4 T4 T4 T4 T4 T4 T4 T4 T4 T4 T4 T4 T4 T4 T4 T4 T4 T4 T4 T4 T4 T4 T4 T4 T4 T4 T4 T4 T4 T4 T4 T4 T4 T4 T4 T4 T4 T4 T4 T4 T4  G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G  1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5  161558031 186283205 196656854 206753691 242997307 41662 21930354 47000363 51035350 83394849 103819868 128716306 131247060 145673807 162918102 210136808 211840216 218406423 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T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53  G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G  8 8 9 9 9 9 9 9 9 9 9 9 9 9 9 10 10 10 10 10 11 11 11 11 11 11 11 11 11 12 12 12 12 12 13 13 13 13 13 13 13 13 13 13 15 15 15  41586944 58157391 37935190 67270003 71333871 77122888 79136921 83221598 90481774 95419404 99552250 105583846 107633128 115724200 127104199 48181069 72583189 76667248 113577597 129620434 11106340 17293901 19248489 60261715 79995579 111537832 114934071 129706418 133980136 6223 1526627 70588953 103377461 121949281 17918001 21089625 23083059 41967454 44927806 45408174 70429098 87156308 98251198 110600643 18272238 21805449 34937367  41975651 58393591 46275010 70788998 72221392 77684326 83098662 89657240 95197578 98300736 103520670 106714988 112014037 126805467 137949835 50621676 73288590 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T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53  G G G G G G G G G G G G G G G G G G G G G G L L L L L L L L L L L L L L L L L L L L L L L L L  15 15 15 15 15 15 16 16 17 17 18 18 18 18 19 20 20 20 20 21 21 22 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2  55550826 58811890 67006694 70693862 71764685 75603709 14634005 46484333 27877766 41103988 32179153 33246557 43778413 70635327 47912047 22146196 28034381 57063146 61845302 13879419 45667514 28845092 617 12724432 17217972 25351676 26465691 27084577 174146398 233378012 25523248 36936339 47752592 54031551 84979184 91608685 98900266 112291685 120082855 128133786 159891003 169863522 176359968 182718234 197782980 201419219 202673686  56641093 59207810 68746547 71172800 72403143 77640945 15414812 46868750 30238813 41357984 32536391 34397042 44723913 71854573 48626174 25235340 28251024 61192343 62435964 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 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53  L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L  3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 6 7 7 7 7 7 7 7 7 8 8 8 8 8 8 9 9 10 10 10 10 10  50670818 139761363 142657253 154054634 170842335 182089134 184091384 196335425 53766377 64081058 67413317 76601966 78196277 89233079 128952512 140187025 151848353 36939489 54439135 61657012 125838199 150080197 159195585 171301660 178692833 76244437 5585965 61087267 64729861 72452483 84446843 94209394 117369099 134704640 22007157 38745689 54928152 95411183 100098076 111875825 35985129 126774264 64411573 69354590 70260473 73239331 126352795  51228685 140244152 143956290 154412636 171665102 183176951 185039705 198168305 54116603 64375660 67604634 77271252 78417632 89804528 129390725 140521672 152580921 37473658 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T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53 T53  L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L  11 11 11 11 11 12 12 12 12 12 12 12 12 12 12 13 13 14 14 14 14 14 14 14 15 15 15 15 15 15 16 16 16 16 17 17 17 17 17 17 17 17 17 18 18 18 18  3573460 69544401 72852680 107109972 131887972 712002 7750091 47207929 47664568 91463507 93408240 111244517 117025244 119676721 131682527 19098344 57621677 20593740 34291680 48748451 57764251 62114710 66358138 80831828 32897394 48336894 56648904 63341491 69961317 91086545 48586222 52778760 56049631 77920768 964480 5120725 24264614 37543886 46400485 53797993 61469371 63118029 64736904 607831 16659756 21843355 23570175  4099388 70008693 73412975 107894392 132151114 1552778 8160827 47466311 48197129 92516893 94098702 111622215 117328738 121965492 132289534 19558501 58001888 21126835 35020664 50329715 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T53 T53 T53 T53 T53 T53 T53 T53 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45  L L L L L L L L L L L L L G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G  18 18 18 19 19 19 19 20 21 22 22 22 22 1 2 2 2 3 3 4 4 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6  25034674 52327118 58393970 11828363 41542240 51436748 53383082 34485436 17603596 16103207 28520754 34376492 35061869 92445707 3214213 35640496 108347119 45246741 109856425 48771496 174746383 17284044 20897033 43267631 45748775 80446929 89734694 102223029 113712200 118943200 68167956 70800143 75184917 84548248 85390617 93357664 95939610 104733720 106006097 108918904 116520215 122111262 129868081 135381781 141913543 145043003 148646458  25284594 52822764 58835881 12483235 43446499 51896236 54181552 35235053 18289926 16594536 28911819 34801814 35449188 92944794 3489907 35914637 109056951 45581671 110182616 49210770 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171 543 109 366 594 736  103  ID  G/L  Chr  Start (bp)  End (bp)  Size  ID  G/L  Chr  Start (bp)  End (bp)  Size  T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45 T45  L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L  2 3 3 3 3 4 5 8 8 8 8 8 8 8 9 10 10 10 10 10 10 10 10 10 10 11 11 11 12 15 15 15 15 16 16 16 16 16 16 16 16 17 17 17 17 17 17  142035883 18708675 75285656 108430837 173127512 3645088 62191844 47102600 78197078 86699540 102139242 105361095 132754059 140178271 82833412 101496919 105427275 108937968 112505977 115390767 117527909 118242031 119109301 123939148 129057100 29591189 96545676 132551773 115722300 18674575 25855573 75603709 92398433 44231 4961896 53615738 60759769 64289064 66600107 69534056 71288486 1882737 7205406 13230426 14696523 17372997 20438273  142422251 18905397 75515882 108693369 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4 4 4 4 4 4 4 5 6 6 7 7 7 7 7 7 7 8 8 8 8 8 8  56063594 74843 9090400 13844286 16184022 32839254 35186754 56430305 60369 815899 29954191 36528233 39677527 44493987 31898817 39766436 167301416 16254527 78394977 140772863 162633116 3645088 6406037 10371953 82568192 115659364 118592935 141100509 142795200 152699346 159111257 93450342 95354742 103346894 24242808 55784611 101876389 108283569 126932281 143515150 153572414 764689 3329536 17007797 19765603 32968313 52621388  76098439 7984147 13472945 15928714 24178465 34943389 56396446 63654245 734327 24411323 35971325 39479846 44130755 61192343 38767124 46927776 167753878 16432161 78583892 141334635 163227623 4065877 7071120 10941759 84598755 116177484 119487920 141832678 148963622 153069035 159988962 93830506 95609774 103604028 24491213 56234811 102207473 108768829 127397229 143839638 153780253 2798932 3634216 19423330 23579445 34237587 53720032  (kb) 200 790 438 208 799 210 212 722 674 235 601 295 445 166 686 716 452 178 189 562 595 421 665 570 203 518 895 732 616 370 878 380 255 257 248 450 331 485 465 324 208 203 305 241 381 126 109  105  ID  G/L  Chr  Start (bp)  End (bp)  Size  ID  G/L  Chr  Start (bp)  End (bp)  Size  T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50  G G G G G G G G G G G G G G G G G G G L L L L L L L L L L L L L L L L L L L L L L L L L L L L  10 10 11 12 13 13 13 13 13 13 13 13 14 14 14 14 15 18 19 1 1 1 1 1 1 2 2 2 2 2 2 2 3 3 3 3 3 3 3 4 4 4 7 7 7 8 8  118147658 128335445 60661723 36302222 20698521 27142461 40460256 43481375 51492647 83509678 89110421 105166636 74831170 89221786 94478987 97332470 50959420 42657928 55554013 3204885 18152027 41399038 47813577 71623262 87291697 5746343 23512042 71611728 134654310 163641233 217405254 238603267 18708675 43774937 53469006 108872278 128108621 129385338 131422673 49189866 77946571 109386058 2864230 57297100 140395448 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10 10 10 11 11 11 11 11 11 12 13 13 13 13 13 13 13 13 13 13 14  57031842 75167545 77458893 86699540 87744061 97285991 103542930 105636430 116012122 133072743 140919525 141840700 1302960 3762511 38500449 82833412 113006323 477678 4260820 13548200 22227856 34351870 43318759 65100222 72339683 103279334 110133250 112801345 117429331 39250444 55463571 84557237 89012345 130977965 132051142 92435964 24483411 27727371 36678499 38419312 46168557 70057505 78513431 90997052 103017055 110362947 98083684  58555824 76418032 78033463 87055819 94497499 98111425 105219912 109065071 126818082 133464258 141422749 143165362 1745895 4605818 38736668 83098662 113222716 3496279 12011805 21627134 32041152 39116217 61160076 66994232 102554000 108738304 110889891 117118223 135279687 39661602 55923540 85007763 89476176 131329903 132389201 92696372 26526090 28177459 37673052 39713370 56269250 73606932 90250492 97825675 108808111 110764162 98569435  (kb) 152 125 575 356 675 825 167 342 108 392 503 132 443 843 236 265 216 301 775 807 981 476 178 189 302 545 757 431 178 411 460 451 464 352 338 260 204 450 995 129 101 354 117 682 579 401 486  106  ID  G/L  Chr  Start (bp)  End (bp)  Size  ID  G/L  Chr  Start (bp)  End (bp)  Size  T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T50 T34 T34 T34 T34 T34 T34 T34 T34 T34  L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L G G G G G G G G G  8 8 9 9 10 10 10 10 10 10 10 13 13 13 16 16 16 16 16 16 16 16 16 16 17 17 17 17 17 17 17 19 20 20 20 22 22 22 1 1 1 1 1 1 1 1 1  8938573 87208452 9067399 82833412 3631141 8851508 48181069 51903179 79776333 130431595 133771689 48069306 49595011 71226333 48106458 49406045 54410314 69643219 71288486 75370384 77371695 77920768 79901920 84535576 2534851 4687532 5418209 9487785 13230426 14415762 17534762 8629145 44493987 56245366 61041281 15930939 16853741 45774295 160338731 162887424 164996988 167684245 173228930 174639051 177945800 179071822 181937518  11424992 87407061 9654318 82953738 4728854 9117982 50237413 52337214 80241593 132473339 134669886 48404010 50888467 71728231 48638074 50618041 55091064 70151844 72036034 75899230 77830699 79572749 82307174 85732272 2937807 5025418 7457682 12620614 14235312 15759518 17865872 8832847 44796823 57461196 61537434 16096955 17206783 47743222 160677252 163728359 167445692 168121001 174053395 177140954 178479748 181045400 183023220  (kb) 2486 199 587 120 1098 266 2056 434 465 2042 898 335 1293 502 532 1212 681 509 748 529 459 1652 2405 1197 403 338 2039 3133 1005 1344 331 204 303 1216 496 166 353 1969 339 841 2449 437 824 2502 534 1974 1086  T24 T24 T24 T24 T24 T24 T24 T24 T24 T24 T24 T24 T24 T24 T24 T24 T24 T24 T24 T24 T17 T17 T17 T17 T17 T17 T17 T17 T17 T17 T17 T17 T17 T17 T17 T17 T17 T17 T17 T17 T17 T17 T17 T17 T11 T11 T11  L L L L L L L L L L L L L L L L L L L L G G G G G G G G G G G G L L L L L L L L L L L L G G G  15 15 15 15 15 15 16 16 16 16 16 16 16 16 16 16 16 16 19 20 4 7 11 11 11 12 12 15 15 15 18 19 2 3 3 7 8 8 8 9 11 11 17 19 1 1 1  24553837 46199533 82472074 85411106 89632877 94546822 44231 15040819 33866283 45548262 47457492 52309608 53615738 61056663 70754647 77175405 78813031 79901920 15874684 39427063 142292357 57163665 60661723 96833936 131887972 36302222 69844305 18674575 30328715 46039586 42657928 23835271 134784717 78394977 140772863 20168369 7035076 136577260 139282254 82833412 79463518 127563703 13230426 8629145 3553533 11435982 16646685  27477714 47809868 85278339 87781613 93718264 100309437 1032612 15414812 35136830 46605357 51434198 52599483 55091064 61730602 73114581 78760401 79773936 82488856 16219285 39747527 142586186 57368235 60880212 97154680 132151114 36432239 70114686 19353919 30597338 46542062 42840984 24178465 135096043 78583892 141053191 20487247 7289755 137000216 140847408 82953738 80194218 128090809 14235312 8832847 3811527 11755347 17086056  (kb) 292 161 280 237 408 576 988 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G G G G G G G G G G G G  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2  24934692 120249993 142533622 145357581 146778005 149747994 152333831 159680000 161016469 163384213 174639051 177945800 182037344 196594358 211445419 214852240 218273469 221391322 223882340 233834184 239216214 242089298 41662 1438693 6648268 14520907 27134515 39691420 41706139 44824074 54554030 58126468 66790913 73109249 84721136 94986258 97249568 101985939 108761390 118601009 132821463 135989619 147842811 148963859 152902602 162516066 175041480  25334144 121077638 145133682 145734554 147083700 151131174 156488812 160677252 161777223 171187941 176344936 181045400 183769234 208088220 213891474 215373858 220609576 221739224 233440847 236328485 241090397 242491480 696156 6391789 12541039 21856943 31254640 40675772 42547144 47789368 55438372 60943837 72007879 74467228 89405301 95863637 98980797 108262692 113816728 120128215 134595150 137582864 148157606 150051708 153748650 163117738 178341522  (kb) 399 828 260 377 306 138 415 997 761 780 170 310 173 114 244 522 233 348 955 249 187 402 654 495 589 733 412 984 841 296 884 281 521 135 468 877 173 627 505 152 177 159 315 108 846 602 330  108  ID  G/L  Chr  Start (bp)  End (bp)  Size  ID  G/L  Chr  Start (bp)  End (bp)  Size  T34 T34 T34 T34 T34 T34 T34 T34 T34 T34 T34 T34 T34 T34 T34 T34 T34 T34 T34 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11  L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L  8 8 8 8 8 8 12 12 12 16 16 16 16 16 16 16 16 16 16 1 1 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 5 5 8 9 9 9  126801789 133072743 134988076 135753903 137364884 143710403 2519891 13450373 36302222 5170141 9148951 14003160 18936858 47811144 48842296 60255176 64289064 69787193 80059470 27020386 190137112 40052595 42380990 43774937 56682157 68278236 69383522 73143638 188713968 193362761 54787033 84575660 97791526 108865061 115659364 125632333 130534517 147902044 173045996 181177641 188522242 29899094 68702646 7035076 23994 937155 1415639  129476219 134244619 135520671 137000216 141332829 143916668 2655195 14142029 36786132 7586817 9954815 14454827 27865580 48264112 56056872 63762681 64876753 79467985 85732272 27234980 190351358 41220493 42652925 45581671 57209359 68785539 70910161 73736485 190600506 195170190 55371595 85692474 98324578 109473917 116742407 125947507 131928006 148530554 174144577 185095200 190387639 30610845 69102023 7289755 964142 1498619 1901823  (kb) 2674 1172 533 1246 3968 206 135 692 484 2417 806 452 8929 453 7215 3508 588 9681 5673 215 214 1168 272 1807 527 507 1527 593 1887 1807 585 1117 533 609 1083 315 1393 629 1099 3918 1865 712 399 255 940 561 486  T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11  G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G  2 2 2 2 2 2 2 2 2 4 4 5 5 5 5 5 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 7 7 7 7 7 7 7 7 7 7 8 10 11 12 13 13  191477009 202017847 207584984 217405254 226834062 229763361 230702740 236163199 237173612 8979402 172753517 1684158 17284044 69602994 95697117 138859931 73242 6231728 11402225 13508273 33017597 45494917 53456669 87028414 89795423 110846002 123929276 149457230 159737023 163376951 167790639 42475 3167180 6038742 11281922 13834918 33604992 57279942 105553799 127398355 143515150 36943 99476175 60661723 52090606 23525346 29471954  192734438 202836206 208489071 220909658 228081471 229961863 234828785 236728809 241063385 9841887 173131406 1965402 17553934 70066836 95852946 139250455 3890080 8075923 11936033 14506540 44166592 46849677 53956516 87408200 91296384 111130824 124385689 149977460 161039688 164236656 168933714 2407575 5446129 8246051 12075944 15851570 34018904 57589990 106086426 127812452 143839638 179853 100248423 60880212 52327387 28177459 33324334  (kb) 125 818 904 350 124 199 412 566 389 862 378 281 270 464 156 391 381 184 534 998 111 135 500 380 150 285 456 520 130 860 114 236 227 220 794 201 414 310 533 414 324 143 772 218 237 465 385  109  ID  G/L  Chr  Start (bp)  End (bp)  Size  T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11  L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L  9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 11 12 12 12 12 13 14 14 14 14 14 14 16 16 16 16 16 16 16 16 17 18 18 19 19 19 22 22  1875733 2271469 2584552 3360032 3627891 4119797 13490884 14568749 16193562 16784842 20133898 31530079 31810164 35779611 39294215 77684451 97278670 32108495 41471084 87593424 113919526 102814184 34291680 35993653 51580716 71515335 73282483 95632850 8317822 12176022 20943307 25365870 29181450 34133153 46597919 60759769 73158376 10976351 23570175 8934066 32423910 55554013 40935061 45618914  2296356 2620657 3399803 3696642 4163314 13011570 14138081 16325485 16784841 17666004 21158464 31868461 33355181 36133758 40762683 78480906 97609941 32334573 41979185 87952085 114162755 103787434 35020664 36534612 52650444 73019214 73691608 97138734 9816450 16391845 22020584 26779984 29392366 35136830 47016802 61286942 73381889 11327785 23897445 9026570 32613567 55707294 41198248 47839586  (kb) 421 349 815 337 535 8892 647 1757 591 881 1025 338 1545 354 1468 796 331 226 508 359 243 973 729 541 1070 1504 409 1506 1499 4216 1077 1414 211 1004 419 527 224 351 327 93 190 153 263 2221  110  ID  G/L  Chr  Start (bp)  End (bp)  Size  T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11 T11  G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G L L L L L L L L L  13 13 13 13 13 13 13 13 13 15 15 15 17 17 17 18 18 19 19 19 19 19 20 20 20 20 20 20 20 21 3 3 3 3 3 3 3 3 3  34965270 39330480 42504729 46168557 50689132 62001010 93396760 102377421 110600643 18674575 45913031 66365409 33264100 41755471 68745974 42657928 62933102 32839254 37053061 44261521 45946143 61364524 60369 7516914 15310602 29954191 32552419 36528233 54011886 21580499 77228270 78394977 114828518 140772863 152957973 156012946 160202103 170074359 182483149  35422901 40124114 43926080 46488557 51467864 62577460 101232430 102837827 114093192 20053874 46223231 66909639 33700089 42239175 69076047 42803873 63153508 36584144 37599399 44608085 49532531 63654245 3341100 11951142 26233197 31357350 35971325 49744292 61192343 21789792 78310037 78583892 115437585 142716241 154220806 156952918 161251745 171101033 183176951  (kb) 458 794 142 320 779 576 783 460 349 137 310 544 436 484 330 146 220 374 546 347 358 229 328 443 109 140 341 132 718 209 108 189 609 194 126 940 105 102 694  Table A.3 ID E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25  aCGH alterations by sample (RB1+/-).  G/L  Chr  Start (bp)  End (bp)  Size  ID  G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G  1 1 4 5 5 6 6 6 8 8 10 10 10 10 10 10 10 10 10 10 10 10 10 11 13 13 13 13 13 13 13 13 13 14 15 15 15 17 17 17 17 17 17 17 17  16638721 232830875 108572846 17284044 171952417 73242 62893855 103346894 58157391 116773589 9174672 13548200 42112266 43318759 45037616 62342913 63597548 70708535 72033929 76667248 78319642 79431219 124978917 46058617 77747248 90250611 92613317 95581707 98714149 100030217 101364859 105535345 108629457 19149713 20588181 27830649 30237319 33264100 54544587 55248203 60576372 62228071 67503237 71406761 74090777  17086056 233105801 108797097 17553934 172226263 346084 63309019 103604028 58393591 117061503 11386923 14520085 43201651 44600038 50711049 63308545 64220266 71438469 73288590 78143315 78833935 85424122 125259149 46407944 78216186 91242980 93325237 98415699 99376982 100888613 102837827 107155648 114093192 19485285 20967788 28937648 30597338 33700089 54973480 55709933 61471405 62594266 69931286 71663462 74851642  (kb) 447 275 224 270 274 273 415 257 236 288 2212 972 1089 1281 5673 966 623 730 1255 1476 514 5993 280 349 469 992 712 2834 663 858 1473 1620 5464 336 380 1107 360 436 429 462 895 366 2428 257 761  T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79  111  G/L G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G  Chr 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 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3  Start (bp)  End (bp)  Size  16646685 120249993 142533622 145511311 147590048 148655012 154247819 158010337 159680000 161016469 162887424 173228930 174639051 177864963 179462543 182037344 199215670 211445419 214172120 217480858 219017537 222704527 224791361 237294682 242294867 246424085 41662 10828736 14520907 27652475 36503134 38942295 39691420 44824074 48847617 50564941 53483127 54256708 58126468 64350737 65474555 83467797 88921446 52725023 155644236  17086056 121077638 144873033 145872489 148144657 153230241 156300507 158487941 160677252 161777223 171525137 174094669 176053350 178479748 181045400 183361276 209169786 213185256 215373858 218795330 220609576 223260778 233391573 239002879 244296856 246944046 9407162 13079174 23881796 31254640 38839346 39196527 43518222 47789368 49273764 51219186 53906745 55105184 60555094 65026194 76133225 88373278 91590359 52967443 156012945  (kb) 439 828 2339 361 555 4575 2053 478 997 761 8638 866 1414 615 1583 1324 9954 1740 1202 1314 1592 556 8600 1708 2002 520 9366 2250 9361 3602 2336 254 3827 2965 426 654 424 848 2429 675 10659 4905 2669 242 369  ID E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E25 E1 E1  G/L  Chr  Start (bp)  End (bp)  Size  ID  G G G G G G G G G G G G G G L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L G G  17 18 18 18 18 18 18 18 18 18 18 19 19 19 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 5 5 5 6 6 8 8 10 11 12 13 17 1 1  78249151 1946123 13210163 26193974 33328621 42226552 53512602 58927478 67604061 70635327 75216360 12856233 15004017 21788967 46140 2804349 22209305 26527404 36350375 42380990 43774937 48536891 51228042 57523638 59349125 63461865 69900482 76936762 86143774 77119217 80523279 82331002 84756808 9117097 161447352 170424700 9303589 163246092 2059659 86699540 477678 121858210 114960307 52275633 56824384 6040269 7778452  78615238 2270736 13643492 26349693 35657079 44723913 54477350 59480384 67966894 74815544 76098439 13472945 16219285 22154696 1857205 18905397 25600762 31335620 41220493 43363544 46115480 50482968 56176682 58715235 63205827 68785539 74025996 78310037 87362355 78634666 81600464 83776911 85692474 9336791 161731582 170635099 9524750 163450786 2279635 87055819 3904258 122274860 115230777 52938369 57089085 6256800 11929541  (kb) 366 325 433 156 2328 2497 965 553 363 4180 882 617 1215 366 1811 16101 3391 4808 4870 983 2341 1946 4949 1192 3857 5324 4126 1373 1219 1515 1077 1446 936 220 284 210 221 205 220 356 3427 417 270 663 265 217 4151  T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79  112  G/L G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G L L L L L L L L L L L L L L  Chr 3 4 4 4 4 4 4 4 5 5 5 6 6 6 6 6 6 8 8 9 10 11 12 12 12 12 15 15 16 16 20 20 20 2 3 3 4 5 5 5 5 5 5 5 5 5 5  Start (bp)  End (bp)  Size  190572724 109386058 140894330 143195412 152158287 160281144 164893174 186022734 17284044 57285917 95697117 73242 10648344 19480476 43851257 44117784 45494917 6515253 58157391 73371288 38983843 60573488 52090606 66859847 108154779 114113748 22796772 28106102 69365103 83933107 26104051 44380338 55869603 91608685 36111427 39737594 132667102 103927370 104995250 106909929 107235949 108502451 112668515 114700474 129057000 134568930 135193500  190793756 109668156 142828372 151471513 159988962 164670201 185959726 191013899 17553934 57537297 95852946 10308635 19088319 43925174 44166592 45175251 58849697 6907559 58393591 73732453 39116217 60880212 52327387 67063558 108581445 114520437 23152527 28734907 69777653 84359546 26233197 44472487 56267785 91680249 36427114 39987069 133073640 104469666 107081530 107306804 108154189 112557699 113857732 118294962 134458770 135240151 141196500  (kb) 221 282 1934 8276 7831 4389 21067 4991 270 251 156 10235 8440 24445 315 1057 13355 392 236 361 132 307 237 204 427 407 356 629 413 426 129 92 398 72 316 249 407 542 2086 397 918 4055 1189 3594 5402 671 6003  ID E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1  G/L G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G  Chr 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 5 5 5 5 5 5 5 5 5 5 7 7  Start (bp)  End (bp)  Size  ID  15896191 17239175 21717568 26424669 34892938 39063444 44122639 51342022 108993704 148535332 152524737 200877039 232955277 9985433 24892640 26714284 31916659 55550156 96074237 202673686 203558341 218744125 232967040 241955282 12335374 46943432 49843917 114814923 121682380 126071864 139855931 170977548 198952699 3705279 38175656 350724 10266635 36939489 53205455 68311858 74566167 86471625 111925202 126810683 137318181 71800030 72809108  16620917 17648568 24615446 29453636 36369397 41233201 45325761 53007168 110039385 149813049 155119104 201431856 233546093 11150862 25572843 28097537 33205438 55858798 96378723 203037744 204180576 219110147 233991801 242720443 12780009 48164871 50482968 115059605 121964439 126244947 140244152 171510249 199276959 4065877 40511826 1562826 10636898 37473658 53387793 69037279 74865527 86737136 112249276 127081020 138329154 72164907 73534009  (kb) 725 409 2898 3029 1476 2170 1203 1665 1046 1278 2594 555 591 1165 680 1383 1289 309 304 364 622 366 1025 765 445 1221 639 245 282 173 388 533 324 361 2336 1212 370 534 182 725 299 266 324 270 1011 365 725  T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T79 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48  113  G/L L L L L L L L L L L L L L L L G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G  Chr 5 5 5 5 5 5 5 5 7 8 11 13 13 20 20 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3  Start (bp)  End (bp)  Size  141348434 155766667 162634259 165508395 167024174 167802449 168095482 168704419 72652466 86558071 97278670 46168557 47429131 29297109 49568098 143900677 157848491 218033276 219229659 230214952 41662 640765 1438693 2815042 11750125 12928689 16361494 18946602 30764067 36371522 59913866 65106400 70345668 86933421 109942316 119008111 120626425 128716306 132821463 138967268 164156982 199032194 216927309 219925081 236568755 26305164 106734450  155252853 161647844 165246970 167079037 167819073 168220953 168709547 179838343 72877032 87055819 97609941 46488557 47613957 29909342 49715808 144162829 158487941 218461661 219532748 230585021 317294 1218689 2663054 8825151 12541039 15892772 18347973 23881796 31915204 36996317 60555094 67279330 72279682 91680249 114143148 119748627 121633928 131497926 134595150 139369283 164779204 199519884 218904083 222928085 241063385 26651493 106950425  (kb) 13904 5881 2613 1571 795 419 614 11134 225 498 331 320 185 612 148 262 639 428 303 370 276 578 1224 6010 791 2964 1986 4935 1151 625 641 2173 1934 4747 4201 741 1008 2782 1774 402 622 488 1977 3003 4495 346 216  ID E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1  G/L G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G  Chr 7 8 8 8 9 9 9 9 9 9 10 10 11 11 11 11 11 11 11 11 11 11 12 12 12 12 12 12 12 13 13 13 13 13 13 14 14 14 15 15 15 16 16 16 16 16 17  Start (bp)  End (bp)  Size  ID  99873579 22867983 67828684 103387755 15176811 20392848 96096087 113177037 123326445 128732487 21655748 104813634 50991 3573460 6246775 46429333 47710156 64048806 72553832 76815285 82399450 118055345 7589386 47274158 54296170 108541669 114851293 121469696 131869058 29951709 42627655 54780783 90478077 97277801 112897047 30308385 72454326 102640563 20302216 73336790 91086545 1718470 29306982 30539947 65200179 88032006 27213580  101989129 23232427 68608798 103959662 15509560 20736915 98512120 114149576 128420787 140185705 22103168 105172292 651922 4099388 6580309 47629247 48122594 67116241 73412975 77212623 82760983 118758082 8010390 49849518 55617585 110713626 115132869 124234371 132289534 30486781 42831764 55110752 90828221 99546821 113901932 31059479 73575089 103624847 20792135 73793805 91292369 2508230 29880154 31120743 66485262 88573088 28028278  (kb) 2116 364 780 572 333 344 2416 973 5094 11453 447 359 601 526 334 1200 412 3067 859 397 362 703 421 2575 1321 2172 282 2765 420 535 204 330 350 2269 1005 751 1121 984 490 457 206 790 573 581 1285 541 815  T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48  114  G/L G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G  Chr 3 3 4 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 6 6 7 7 8 8 8 9 10 11 11 12 12 13 13 13 13 13 14 15 15 15 16  Start (bp)  End (bp)  Size  108016085 134944834 48771496 70263 7664722 10597609 17284044 20897033 21907045 32789563 37446811 50797452 55064491 55718971 56587456 58183784 65479269 68951921 72433622 72933899 75354258 76889115 79041719 180195851 73242 106731286 57163665 102001310 6515253 58157391 86196327 45908108 50596911 118980810 131887972 36090539 111588013 63561919 90907034 101364859 102894434 110362947 103570986 18272238 19087319 30237319 31814669  108230341 135140571 49416673 7150950 10140637 16847656 18012097 21549907 22929763 36158483 43279969 54459262 55411390 56366596 57537297 61656696 68369694 72139981 72928146 75429647 75669523 77472799 79525328 180444136 20763610 106914529 57368235 102640752 6907559 58393591 86435637 46458217 50869761 119462934 132151114 36311121 112250545 64068098 101083925 102690682 109977945 113901932 103750538 18717253 20224003 30597338 32428987  (kb) 214 196 645 7081 2476 6250 728 653 1023 3369 5833 3662 347 648 950 3473 2890 3188 495 2496 315 584 484 248 20690 183 205 639 392 236 239 550 273 482 263 221 663 506 10177 1326 7084 3539 180 445 1137 360 614  ID E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1  G/L G G G G G G G G G G G G G G G G G G G G G G G G G G G L L L L L L L L L L L L L L L L L L L L  Chr 17 17 17 17 18 18 18 18 19 19 20 20 20 20 20 20 20 20 20 21 21 21 22 22 22 22 22 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 4 4 4 4 4  Start (bp)  End (bp)  Size  ID  30462926 50943878 65852467 73330751 16897445 18745629 25034674 62103149 18013971 54795121 2658810 5506268 29486920 30430011 45219566 48069170 51597084 54316042 61386952 20794703 36306335 46391179 17475778 19054378 28126621 30419219 35061869 714656 1438693 3214213 60112709 76560762 94689898 131538597 158728287 180385399 35484750 80918434 97715503 163846727 166701905 190572982 22846486 65613252 68980297 76015896 78040557  45085833 63366540 73230854 78615238 18303681 19695229 25284594 62837383 19781144 55203857 4125081 6145804 29909342 34853598 45756815 50228867 51966817 54747849 62435964 21052440 36850123 46927776 18549230 19736241 30166633 30768051 41661864 944883 1649373 3489907 60403012 77347435 95017226 131894005 159062951 180842344 36234563 81176295 98211952 164196155 167210047 190902546 23104480 65926998 70337363 76544458 78823975  (kb) 14623 12423 7378 5284 1406 950 250 734 1767 409 1466 640 422 4424 537 2160 370 432 1049 258 544 537 1073 682 2040 349 6600 230 211 276 290 787 327 355 335 457 750 258 496 349 508 330 258 314 1357 529 783  T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48  115  G/L G G G G G G G G G G G G G G G G G L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L  Chr 16 16 18 19 19 19 19 19 19 19 19 19 19 19 20 20 20 1 1 1 1 1 1 1 1 1 1 1 1 1 3 3 4 4 4 4 4 5 5 5 5 6 6 6 6 6 6  Start (bp)  End (bp)  Size  33400710 59038955 42657928 12587922 15264050 32839254 35186754 39679939 43448022 46693651 47784067 50398622 55707295 59799647 60369 28034381 32552419 42399181 47658247 59931165 62007293 63017988 68379704 74579113 77685641 90930813 99921386 106804596 108214875 117081066 129036625 197279380 39007380 64081058 113777516 128952512 151848353 75247038 95697117 172799092 176300084 20728596 63255599 73442278 84456091 89365634 93274524  33693171 59260726 42840984 13472945 16168320 34943389 37230471 40136267 43949819 47430252 48626174 50652469 56396446 62794248 26233197 31357350 62435964 47483542 59669503 60603939 62705908 68211732 69138390 76344381 90021480 95323264 104094805 107453098 116827699 120078146 129292335 198168305 39806180 64375660 114063996 129390725 152080415 75421795 95852946 173016342 176701325 58849697 72853784 79116376 89259710 92119551 93727522  (kb) 292 222 183 885 904 2104 2044 456 502 737 842 254 689 2995 26173 3323 29884 5084 12011 673 699 5194 759 1765 12336 4392 4173 649 8613 2997 256 889 799 295 286 438 232 175 156 217 401 38121 9598 5674 4804 2754 453  ID E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E1  G/L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L  Chr 4 5 5 5 6 6 6 6 6 6 7 7 7 7 8 8 8 9 9 9 10 10 10 10 10 10 10 10 10 10 11 11 12 13 13 13 13 13 15 15 15 15 15 15 16 17 17  Start (bp)  End (bp)  Size  ID  142292357 95697117 99124137 170231354 58352741 62000083 73442278 85390617 149900596 160616018 10936621 48534377 66502015 81155139 85434907 86699540 132297428 43111687 67270003 67803916 4260820 48181069 76667248 86396539 89987583 95583121 108801518 119281891 124978917 131945177 15478026 47479617 38427897 49768827 52402856 56242491 71226333 72702354 18272238 20762791 21805449 34634629 37191685 55550826 33730527 2938114 16205935  142686744 95992566 99378879 170505998 58849697 62977588 73844143 85796999 150732866 161039688 11842183 48933660 66754917 81349702 85886119 87055819 132772696 44458663 67409630 70224724 5235474 49216977 78484077 88170035 90397121 96037340 109288132 120267795 125357696 133477631 15810241 47842883 38702944 51467864 53318542 57788215 72149680 74860115 18543505 21207304 22655917 34933419 37846674 56126615 34285558 3371434 17035446  (kb) 394 295 255 275 497 978 402 406 832 424 906 399 253 195 451 356 475 1347 140 2421 975 1036 1817 1773 410 454 487 986 379 1532 332 363 275 1699 916 1546 923 2158 271 445 850 299 655 576 555 433 830  T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T48 T19 T19 T19 T19 T19 T19 T19 T19  116  G/L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L G G G G G G G G  Chr 6 6 6 6 6 6 7 7 7 7 8 8 9 9 9 11 11 11 12 12 12 13 13 13 14 14 14 14 14 15 15 15 15 16 16 17 17 17 19 1 1 1 1 1 1 1 1  Start (bp)  End (bp)  Size  95000985 105835894 106899458 150084499 150414015 159443190 5691553 64207804 68323685 129173245 42528859 74696386 19026977 36214616 138571659 66670511 72852680 132728882 7589386 110370511 117025244 43986021 69889347 86479579 34291680 35993653 44520774 49100288 63765831 20302216 48336894 62141088 91086545 18750161 51601593 55205410 59751854 62517580 32504109 3553533 27020386 120249993 142533622 144594746 145584161 150229090 155029936  101234842 106319178 149977460 150405281 158417956 170694605 6688617 64485547 68727422 129771659 42880975 75318841 19373997 36774290 139070516 67116241 73412975 133121207 8160827 111395046 117328738 63637946 81142276 90923076 35777003 38968001 45497527 49350925 64018070 20627698 49146558 62552268 91292369 19546524 51928256 56647031 60166708 63701032 32613567 3811527 27234980 121077638 144162829 145133682 147083700 153230241 158487941  (kb) 6234 483 43078 321 8004 11251 997 278 404 598 352 622 347 560 499 446 560 392 571 1025 303 19652 11253 4443 1485 2974 977 251 252 325 810 411 206 796 327 1442 415 1183 109 258 215 828 1629 539 1500 3001 3458  ID E1 E1 E1 E1 E1 E1 E1 E1 E1 E1 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3  G/L L L L L L L L L L L G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G  Chr 17 17 17 18 18 19 19 19 22 22 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 3 3 3 3 3 5 5 5 5  Start (bp)  End (bp)  Size  ID  21762537 22287134 73158376 15026396 61756829 47784067 50398622 55138786 17053965 18401409 28120980 29953410 39063444 44687143 52663511 151476033 176574921 177764373 179640235 181937518 199539859 204625506 205931802 209299053 222335221 223536766 230214952 232727671 241399361 242089298 245436183 8295224 9985433 24892640 26714284 31916659 55550156 96151128 9232089 49081051 51996266 170977548 198952699 56264214 85422174 137318181 175623077  22154806 22595733 73381889 15320434 62138970 48626174 50652469 55346714 17206783 19036350 29453636 30162031 40504667 45130859 53007168 155119104 176971757 178479748 179933730 182204185 203475222 204975414 206903358 211282305 223152230 224717804 231685446 233546093 241887674 242642629 245747506 9048252 11025497 25572843 27037976 33205438 55858798 96842993 9986408 50121242 52619044 171510249 199276959 56679342 85713517 137920380 176701325  (kb) 392 309 224 294 382 842 254 208 153 635 1333 209 1441 444 344 3643 397 715 293 267 3935 350 972 1983 817 1181 1470 818 488 553 311 753 1040 680 324 1289 309 692 754 1040 623 533 324 415 291 602 1078  T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19  117  G/L G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G  Chr 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 5 5 5 5 6 6 6  Start (bp)  End (bp)  Size  160338731 161016469 170814706 174639051 179462543 180659732 188010203 199091008 205282499 211445419 216039670 218187008 219017537 222704527 224861037 242089298 41662 714656 2815042 9512580 13269911 14137447 26714284 27515896 30019511 36371522 41706139 44563993 48847617 54031551 57719211 58638970 63994493 68416702 70345668 73670503 83467797 88921446 3179266 130702173 17284044 20897033 59689279 86146718 73242 662233 26395979  160677252 167445692 171525137 176344936 180447764 181045400 188453331 204975414 208088220 215373858 217850157 218795330 220609576 223064465 233105801 242491480 317294 2663054 8825151 13154386 13575685 23881796 27037976 29595165 31254640 41105817 42547144 47697639 51219186 55438372 58137159 61582975 67943236 69154160 73168364 82010715 88331961 91680249 3667769 131070392 17553934 21399027 59973593 86407148 346084 26126575 44815780  (kb) 339 6429 710 1706 985 386 443 5884 2806 3928 1810 608 1592 360 8245 402 276 1948 6010 3642 306 9744 324 2079 1235 4734 841 3134 2372 1407 418 2944 3949 737 2823 8340 4864 2759 489 368 270 502 284 260 273 25464 18420  ID E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3  G/L G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G  Chr 6 6 6 6 6 6 6 6 6 6 6 7 7 7 7 7 7 8 9 9 9 9 10 11 11 11 11 11 11 11 12 12 12 14 14 15 15 16 16 17 17 17 17 17 17 17 17  Start (bp)  End (bp)  Size  ID  73242 4392553 6362577 13508273 19867614 24229108 29457137 32872724 39096919 39928065 90402399 2038803 4719283 66605756 72809108 98657508 129173245 144602260 19026977 113177037 125895769 128732487 126236132 50991 10327230 47194406 57164193 61823795 65808280 107109972 5841370 55654146 121469696 73682195 98532350 57254835 72441859 3800374 54179429 510963 39961732 43286090 57842004 69769995 73330751 75566340 76498848  3553636 5437064 7834700 18231811 23016737 29123783 32151955 38315288 39555660 44642460 90731676 2407575 6688617 67227917 73534009 102036700 129934305 146208093 19373997 113848887 128420787 132999756 126557171 2372103 10641310 47629247 57354260 62515502 67116241 107646360 7045503 56198614 122996454 74277120 100389009 57757359 73217136 4125558 54393514 1432425 41722683 46634199 58351860 73230854 73695161 75862414 78615238  (kb) 3480 1045 1472 4724 3149 4895 2695 5443 459 4714 329 369 1969 622 725 3379 761 1606 347 672 2525 4267 321 2321 314 435 190 692 1308 536 1204 544 1527 595 1857 503 775 325 214 921 1761 3348 510 3461 364 296 2116  T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T19 T9 T9 T9 T9 T9 T9 T9 T9 T9 T9 T9  118  G/L G G G G G G G G G G G G G G G G G G G G G G G G G L L L L L L L L L L L G G G L L L L L L L L  Chr 6 6 6 7 8 8 11 14 14 14 14 14 14 14 14 14 14 14 15 15 15 16 16 19 19 1 3 5 5 11 13 13 13 13 13 16 6 11 19 4 4 5 5 5 5 5 5  Start (bp)  End (bp)  Size  45632263 46547724 58230371 76182168 11914818 58157391 60661723 51180359 54110331 60182950 67008179 74831170 83363672 86623012 88345764 94478987 97754378 105264727 18473375 20588181 30006525 6091158 33280312 8031324 59799647 100166580 175445960 139059617 180195851 132728882 47570684 52275633 52597405 58025605 76689469 79667658 73242 60661723 55554013 3362479 190667654 55718971 60902209 62347735 67361489 70983047 72433622  46448077 56803717 58849697 76393428 12350857 58393591 60880212 51608095 54510436 62987077 68444627 82992497 85291082 87817262 94182499 97482889 98569435 106318374 19917319 20967788 30539910 6591299 33584068 8832847 59983377 100520827 175738257 139415354 180444136 133121207 48404010 52741383 52938369 58861408 77764260 79901524 346084 60880212 55707294 3610639 190953845 56021821 62085860 62698196 67899255 72029339 72680299  (kb) 816 10256 619 211 436 236 218 428 400 2804 1436 8161 1927 1194 5837 3004 815 1054 1444 380 533 500 304 802 184 354 292 356 248 392 833 466 341 836 1075 234 273 218 153 248 286 303 1184 350 538 1046 247  ID E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3  G/L G G G G G G G G G G G G G G G L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L  Chr 18 19 19 19 19 19 19 20 20 21 22 22 22 22 22 1 1 2 2 2 2 2 2 2 3 3 3 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 6 6 6 6 6  Start (bp)  End (bp)  Size  25034674 1331124 10430640 16184022 43206161 50652470 54795121 43775966 48069170 20794703 15930939 17053965 26434947 35236505 41150627 60771032 246675462 25523248 77054044 96783694 131538597 161044707 193284705 195224267 3791057 88845493 138266982 19191361 22846486 78267102 84891357 96537113 97896455 118973543 132667102 49479231 50839218 75006893 100253676 104100507 125401591 180093032 74510677 77454026 86423400 160616018 170655612  25284594 5186670 13371577 18306025 43719482 54706941 55203857 44472487 48483551 21052440 16096955 17206783 26747513 41130527 42198659 61104212 246944046 25753042 77302660 97695429 131894005 161442145 193703377 195907912 4401633 90244571 139234445 19814983 23104480 78823975 85692474 97073537 98480811 119393936 133073640 50093939 51198402 75588705 100665725 105046456 125696404 180343927 75644516 77857011 87841029 161039688 170880179  (kb) 250 3856 2941 2122 513 4054 409 697 414 258 166 153 313 5894 1048 333 269 230 249 912 355 397 419 684 611 1399 967 624 258 557 801 536 584 420 407 615 359 582 412 946 295 251 1134 403 1418 424 225  ID T9 T9 T9 T9 T9 T9 T9 T9 T9 T9 T9 T9 T9 T9 T9 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1  119  G/L L L L L L L L L L L L L L L L G G G G G G G G G G G G G G G G G G G G G G G G G G G G L L L L  Chr 5 5 5 5 5 5 7 7 8 13 13 13 13 14 18 1 1 1 1 3 3 3 4 4 4 5 5 5 5 6 8 11 11 11 11 12 12 15 16 17 17 18 18 1 1 1 2  Start (bp)  End (bp)  Size  72933899 75354258 81524522 88445542 95032442 106882458 115766919 156780334 135551492 39236837 40912907 42627655 42832083 105434666 75216360 73684664 143900677 160735297 246424085 121556090 139398053 182708944 48771496 84397159 144726984 17284044 25632061 75420049 138859931 104973405 78915837 60661723 61361854 77911332 96545676 67574005 80125405 26199536 21939143 5120725 54544587 11863172 57821908 34421604 222704527 243262497 6858150  73837320 75588705 81697072 88893940 95791871 107135305 115974020 157782629 135765484 39713370 42694359 42831764 43127842 105845800 75531003 73883486 144162829 161015720 246741718 121848103 139636565 183039921 49210770 84777340 144928389 17553934 25955183 75750313 139105069 105325375 79567870 60880212 61860041 78206818 96833884 67904215 80564119 26597300 22187488 5420944 54877132 12316092 58047654 34697333 222960993 243466817 7080915  (kb) 903 234 173 448 759 253 207 1002 214 477 1781 204 296 411 315 199 262 280 318 292 239 331 439 380 201 270 323 330 245 352 652 218 498 295 288 330 439 398 248 300 333 453 226 276 256 204 223  ID E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E3 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5  G/L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L G G G G G G G G G G G G  Chr 7 8 8 8 8 8 8 9 9 10 10 11 11 11 11 11 12 12 13 13 14 14 14 15 15 16 16 17 17 17 17 18 18 19 19 1 2 2 2 2 2 2 2 2 2 2 2  Start (bp)  End (bp)  Size  48534377 43185803 47568877 60811916 62881808 72706992 86699540 11531920 120758160 32785206 100371823 29516501 48090650 50230022 62532845 93414925 38427897 77871943 51074984 54559086 26520749 38967497 44763469 34634629 79676203 33400710 52550335 21762537 22287134 68744962 73158376 15026396 18385344 23835271 50398622 190957775 1642610 3293984 3903894 9249270 23512042 24892640 26714284 28214990 30764067 42689036 73898940  48903100 43912178 47901818 61320284 63455889 73494710 87055819 11963059 121059699 33316028 100906136 29810959 48335007 51421925 62994414 93936247 38702944 78081993 51467864 55373857 27084469 39556557 45817331 34933419 79953718 34490212 52874597 22154806 22595733 69076047 73381889 15320434 18758389 24178465 50652469 191386589 2663054 3748630 4615940 11931175 23881796 25450109 27284933 28756000 31254640 43220834 74792844  (kb) 369 726 333 508 574 788 356 431 302 531 534 294 244 1192 462 521 275 210 393 815 564 589 1054 299 278 1090 324 392 309 331 224 294 373 343 254 429 1020 455 712 2682 370 557 571 541 491 532 894  ID T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1  120  G/L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L  Chr 2 2 2 2 2 3 3 3 3 3 3 4 4 5 5 5 5 5 5 6 6 6 6 6 6 6 6 7 7 8 8 9 9 10 10 10 12 12 12 12 12 12 13 13 13 13 13  Start (bp)  End (bp)  Size  60553094 124195582 157294604 186879569 205347096 20821246 78394977 105928983 124714733 127309873 140668983 13423 182808800 197527 12839471 55920336 72433622 93017881 98042952 42123312 75466376 78146017 79822171 93016222 123300422 143011827 159589768 52841765 154922742 8242248 69645093 68131743 120873426 29448208 116116186 118147658 6511402 49598089 86487712 95788252 99185936 114851293 42903254 46289605 46386689 47901160 50026338  61048014 124430093 157467503 187224688 205689005 21090936 78583892 106173765 125016814 127565087 141334635 256033 183027837 555524 13174647 56144127 72680299 93284274 98364209 42315751 76023862 78314751 80031136 93431583 123547214 143259770 159892720 53284451 155128400 8571704 69999863 68460734 121059699 29797600 116299949 118360397 6754179 49987763 86754944 96085223 99487301 115230777 43127842 46518008 47441575 48242971 51042932  (kb) 495 235 173 345 342 270 189 245 302 255 666 243 219 358 335 224 247 266 321 192 557 169 209 415 247 248 303 443 206 329 355 329 186 349 184 213 243 390 267 297 301 379 225 228 1055 342 1017  ID E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5  G/L G G G G G G G G G G G G G G G G G G G G G G G G G G G L L L L L L L L L L L L L L L L L L L L  Chr 2 2 2 3 5 6 6 7 7 9 10 13 13 15 15 15 17 17 18 18 18 18 18 18 19 20 21 1 1 1 1 1 1 1 1 2 3 3 3 3 4 4 4 4 4 4 4  Start (bp)  End (bp)  Size  ID  79806185 87990794 185880300 9512372 17284044 43995870 154129550 30863988 64972395 138457104 103733984 93396760 108359831 19087319 30237319 72889509 27213580 77390101 35421 5467960 16793577 18745629 21543717 75216360 10216515 42815688 43899532 1058909 5415955 12724432 55481887 59319690 60771032 67900421 92317925 25382449 78394977 81954578 126980939 190572724 764772 9608055 23247592 25775550 28130866 35485307 45905935  80083233 88803463 186157143 9986408 17554876 44268248 154379516 31319741 65209849 138919624 104069156 102958785 113901932 20097557 30597338 73217136 27812924 78036762 3100070 15060997 18073471 21117291 22489038 76098439 10436184 43159048 44194069 1487588 5868138 13569886 56411469 59815749 61339873 69138390 92612882 25753042 78583892 82288232 127282195 190902546 1020003 19814983 23876872 27257262 29198517 37370613 47129172  (kb) 277 813 277 474 271 272 250 456 237 463 335 9562 5542 1010 360 328 599 647 3065 9593 1280 2372 945 882 220 343 295 429 452 845 930 496 569 1238 295 371 189 334 301 330 255 10207 629 1482 1068 1885 1223  T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T1 T76 T76 T76 T76 T76 T76 T76 T76 T76 T76 T76 T76 T76 T76 T76 T76 T76 T76 T76 T76 T76 T76 T76 T76 T76 T76 T76 T76 T76 T72 T72 T72  121  G/L  Chr  Start (bp)  End (bp)  Size  L L L L L L L L L L L L L L L L L L L L L L L L L L L L L G G G G G G G G G G G G G G G G G G  13 13 13 13 15 15 15 16 16 16 16 16 18 18 19 6 2 5 6 8 12 13 13 13 13 13 13 15 19 1 1 1 1 5 5 5 5 9 9 13 13 13 13 15 1 1 1  52541033 59352443 63233263 109502184 20217252 21290128 73769351 51601593 53058404 65720643 68343618 79901920 25034674 69281624 7749446 13223270 211144204 11106805 151049955 86699540 45789177 35996176 36816516 43481375 45408174 67175227 76862480 19884809 32504109 16646685 143665914 177293770 232830875 4280335 17284044 62919710 68905030 122514480 124184461 50689132 66207174 94004743 96341247 30237319 16646685 143219071 155801127  53095154 59638075 63637946 109838498 20338507 21703185 74357465 51928256 53364181 66067662 68959796 80284468 25284594 69613151 7984147 13524298 211423562 11366567 151280704 87055819 46073952 36510125 37223607 44009817 45768530 67681892 77225206 20071155 32613567 17086056 144162829 177908342 233105801 5240403 17553934 63197515 70342024 123029731 124491178 51467864 66805219 95306078 96782355 30597338 17086056 144162829 156042705  (kb) 554 286 405 336 121 413 588 327 306 347 616 383 250 332 235 301 279 260 231 356 285 514 407 528 360 507 363 186 109 439 497 615 275 960 270 278 1437 515 307 779 598 1301 441 360 439 944 242  ID E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5 E5  G/L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L  Chr 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 6 7 7 7 8 8 8 8 8 8 8 8 8 8 9 9 9 9 9 9 9 10 12 12 12 13 13 13 14 14 16  Start (bp)  End (bp)  Size  ID  52354875 55204895 67582165 73869033 78343948 82738643 91158111 104154615 108540535 111625951 119871532 137303256 139331093 145066326 181177641 174556369 13223270 24242808 88158926 156780334 4038043 43422554 46969582 48324637 49937784 72706992 76656394 86699540 117853642 131773301 2271469 8787144 18454415 42064305 109623395 118654542 121713094 41947396 29727999 37754701 59227752 61014574 62395584 64861982 38847479 80635044 15040819  54561183 55856179 70337363 76067534 80379354 83530160 91923787 104907229 108797097 111926554 121956556 137853745 140107223 147630673 182980569 174812270 13577224 24445001 88740787 157071420 4938784 43912178 48171893 48787064 50475395 74263376 77072172 87055819 120752324 132059478 3399803 9654318 18875253 46458217 110355803 118945140 122361130 42224061 29929154 38138583 59583586 62048015 64544139 65274978 39263310 80879562 15407666  (kb) 2206 651 2755 2199 2035 792 766 753 257 301 2085 550 776 2564 1803 256 354 202 582 291 901 490 1202 462 538 1556 416 356 2899 286 1128 867 421 4394 732 291 648 277 201 384 356 1033 2149 413 416 245 367  T72 T72 T72 T72 T72 T72 T72 T72 T72 T72 T72 T72 T72 T72 T72 T72 T72 T72 T72 T72 T72 T72 T72 T72 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20  122  G/L G G G G G G G G G G G G L L L L L L L L L L L L G G G G G G L L L L L L L L L L L L L L L L L  Chr 1 5 7 11 11 13 13 14 15 15 19 22 2 3 3 6 7 8 11 12 14 18 19 22 17 17 17 18 20 20 1 1 2 2 2 2 2 2 3 3 3 3 4 4 4 4 4  Start (bp)  End (bp)  Size  232830875 20897033 57163665 118980810 132051142 108629457 110600643 19149713 21973970 22796772 8629145 14410042 91608685 78394977 114828518 160616018 77228817 7035076 97278670 32108495 72215467 12806854 32504109 40935061 54447833 61469371 67376846 42657928 692082 52483586 44277602 217124947 96785607 120401666 140828781 143058946 169824122 226758143 24994819 36111427 37703118 78394977 3645088 29816475 62298232 104719821 138014420  233105801 21399027 57368235 119354299 132389201 109102920 112948798 19485285 22386839 23152527 8784792 14660949 91680249 78583892 115117759 161039688 77535943 7289755 97609941 32334573 72636951 13135104 32613567 41198248 54973480 61961765 69931286 42803873 1452295 52791277 44370880 217262659 97054766 120706743 141251152 143562739 170250237 227038326 25264504 36427114 37973246 78583892 4065877 30070593 62743878 105038688 138600123  (kb) 275 502 205 373 338 473 2348 336 413 356 156 251 72 189 289 424 307 255 331 226 421 328 109 263 526 492 2554 146 760 308 93 138 269 305 422 504 426 280 270 316 270 189 421 254 446 319 586  ID E5 E5 E5 E5 E5 E5 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20  G/L L L L L L L G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G  Chr 16 16 16 17 17 19 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 3 4 4 4 4 5 5 5  Start (bp)  End (bp)  Size  ID  29181450 52550335 77920768 22287134 68744962 50398622 16646685 62133505 92776245 142533622 148925197 154247819 156649526 161016469 162887424 170587093 173228930 174639051 176574921 179462543 186076660 195455701 198317928 217329398 224861037 234814970 237944944 243223806 925973 5764382 11604683 12928689 16361494 28067510 29024702 36503134 72794626 165764127 234482908 49579895 8617326 132469344 139960125 179461534 12637462 17277342 19832654  29392366 52874597 79358404 22595733 69076047 50652469 17086056 62475327 93254333 145872489 153230241 155835711 160677252 161777223 167842836 171187941 174094669 176344936 178811659 181831357 186470859 196149918 215373858 220609576 233105801 236029274 241203475 245038844 1218689 9407162 12102751 13348548 16914958 28504340 31254640 36996317 73168364 165980904 234828785 49782857 8780507 132802100 140321282 179886545 13007203 17553934 20176670  (kb) 211 324 1438 309 331 254 439 342 478 3339 4305 1588 4028 761 4955 601 866 1706 2237 2369 394 694 17056 3280 8245 1214 3259 1815 293 3643 498 420 553 437 2230 493 374 217 346 203 163 333 361 425 370 277 344  T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20  123  G/L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L  Chr 5 5 5 5 5 5 6 7 7 7 7 7 8 8 8 9 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 11 11 12 13 13 13 13 13 13 13 13 13 13 13 13  Start (bp)  End (bp)  Size  22506952 27140678 49479231 75160802 91292937 93244813 151040678 1499748 61087267 84446843 118629304 145131434 85161425 86558071 120992957 128256342 38983843 41947396 47928933 48969280 50519291 54718352 60249701 60520851 90654816 98960137 108937968 115215602 115994387 119422575 127834059 128961549 25239852 100820386 85650592 19646778 29471954 41563363 42472202 43481375 46386689 53430317 57621677 64966450 68841601 69468748 70791339  22833632 27548165 49636937 75421795 91664641 93709268 151280704 1737551 61797715 84898403 119269525 145947929 85626176 87055819 122162585 128682074 39116217 48053466 49013085 50458757 54498532 58201767 60552579 89428545 98693465 108738304 114878030 115832934 118749875 127882581 128595500 135279687 25489221 101077028 86068176 20414099 29739222 42494518 43127842 43792169 47441575 55655559 64356550 68595839 69301983 70603880 81289277  (kb) 327 407 158 261 372 464 240 238 710 452 640 816 465 498 1170 426 132 6106 1084 1489 3979 3483 303 28908 8039 9778 5940 617 2755 8460 761 6318 249 257 418 767 267 931 656 311 1055 2225 6735 3629 460 1135 10498  ID T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20  G/L G G G G G G G G G G G G G G G G G G G G G G G G G G G G  Chr 5 5 6 7 8 9 9 9 9 10 10 10 10 11 11 12 12 14 15 15 15 15 16 16 16 17 17 17  Start (bp)  End (bp)  Size  49960569 106909929 73242 117127748 80712778 33336233 35583889 131137899 133976381 75055 6112562 13630717 20159003 18978846 60508638 53976376 71994675 19149713 21973970 28106102 34937367 92651955 33280312 47189283 72964202 44818672 47498136 51913051  50290883 107306804 346084 117527587 81019111 33775694 36033505 131452656 134847756 5795590 11008217 14736903 21384213 19286458 60880212 54173283 72180449 19360529 23152527 28734907 35376632 93031016 33741546 47753661 73144686 45685773 47977547 53110374  (kb) 330 397 273 400 306 439 450 315 871 5721 4896 1106 1225 308 372 197 186 211 1179 629 439 379 461 564 180 867 479 1197  ID T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20 T20  124  G/L L L L L L L L L L L L L L L L L L L L L L L L L L L  Chr 13 13 13 13 13 13 13 15 15 16 16 17 17 17 17 17 18 19 19 19 19 19 19 21 21 22  Start (bp)  End (bp)  Size  83509678 84556448 87575870 94351464 102618440 105299403 106405817 19884809 99860521 33659117 79901920 2442274 9979758 13230426 14560485 17656740 12806854 8934066 32504109 36871853 41542240 55554013 58250784 20794721 36306335 40935061  84537312 87198713 94267342 96029356 105229814 106278034 107155648 20071155 100126412 33927835 80284468 3513298 13098904 14103719 15759518 20683824 13135104 9026570 32613567 37397010 41714071 55707294 58500199 21341020 36609093 41198248  (kb) 1028 2642 6691 1678 2611 979 750 186 266 269 383 1071 3119 873 1199 3027 328 93 109 525 172 153 249 546 303 263  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.  125  -1  1  -1  1 -1  1  2 -1  1  -1  -1  -1  4  3  1  1  1  -1  1  5  1  -1  10  -1  7  1  -1  18  1  1  13  -1  1  14  -1  -1  1  1  15  16 1  1 -1  17  -1  8  -1  -1  1  1  12  11  1  6  -1  9  -1  19  1  -1  20  1  -1  21  1  -1  22  -1  1  X  1  Y  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.  126  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 Differential Regulation of Cytokine Production in Macrophages and T Helper Cells by IL-17A and IL-17F  -log(p-value)* 1.68  Ratio 1.11E-01  IL10, IL9  Role of Cytokines in Mediating Communication between Immune Cells Differential Regulation of Cytokine Production in Intestinal Epithelial Cells by IL-17A and IL-17F Glioma Invasiveness Signaling  1.5  5.45E-02  IL10, IL20, IL24  1.48  8.7E-02  IL10, IL9  1.44  5E-02  Leukocyte Extravasation Signaling  1.42  3E-02  CD40 Signaling Maturity Onset Diabetes of Young (MODY) Signaling IL-17A Signaling in Airway Cells Amyotrophic Lateral Sclerosis Signaling NGF Signaling  1.35 1.35  4.29E-02 6.25E-02  PIK3C2B,CD44 (includes EG:100330801), TIMP2 (includes EG:21858) ROCK1, PIK3C2B, EZR, CD44 (includes EG:100330801), RASSF5, TIMP2 (includes EG:21858) PIK3C2B, IKBKE, MAPKAPK2 CACNA1E, CACNA1S  1.32 1.32  4.17E-02 3.36E-02  1.3  3.45E-02  * The –log(p-value) significance threshold for this dataset of 1.30  127  Molecules  PIK3C2B, IL19, IKBKE PIK3C2B, CACNA1E, CACNA1S, GLUL ROCK1, PIK3C2B, NTRK1, IKBKE  Table A.5  Top significantly disrupted canonical pathways in RB1-/- tumors.  Ingenuity Canonical Pathways Antigen Presentation Pathway  -log(pvalue)* 17.4  Ratio  Molecules  5.12E-01  HLA-DMA, PSMB9, HLA-DOA, HLA-DQA1, HLADRB1, HLA-DMB, PSMB8, TAP1, HLA-DPA1, HLA-F, HLA-A, HLA-E, HLA-DRA, HLA-B, HLADOB, HLA-DQA2, HLA-G, HLA-DPB1, TAP2, TAPBP, HLA-DRB5, HLA-C Dendritic Cell 10.8 1.76E-01 FCGR3B, FCGR2C, HLA-DOA, NFKBIE, HLAMaturation 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, HLADOB, IRF8, TNF, HLA-DRB5 Graft-versus-Host 9.92 3.4E-01 HLA-DMA, HLA-DOA, HLA-DQA1, HLA-DRB1, Disease Signaling 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 9.59 2.79E-01 HLA-DMA, HLA-DOA, IL10, HLA-DQA1, HLADisease Signaling 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 8.7 2.19E-01 HLA-DMA, HLA-DOA, IL10, HLA-DQA1, HLASignaling DRB1, HLA-DMB, HLA-DQB1, HLA-DPA1, HLAF, HLA-A, HLA-E, HLA-DRA, HLA-B, FCER1G, HLA-DOB, HLA-DQA2, HLA-G, TNF, HLA-DPB1, HLA-DRB5, HLA-C OX40 Signaling 8.5 2.21E-01 HLA-DMA, HLA-DOA, TNFRSF4, NFKBIE, HLAPathway DQA1, HLA-DRB1, HLA-DMB, HLA-DQB1, HLADPA1, HLA-F, HLA-A, HLA-E, HLA-DRA, HLA-B, FCER1G, HLA-DOB, HLA-DQA2, HLA-G, HLADPB1, HLA-DRB5, HLA-C Type I Diabetes 7.85 1.9E-01 HLA-DMA, HLA-DOA, NFKBIE, HLA-DQA1, Mellitus Signaling HLA-DRB1, HLA-DMB, IKBKE, MAPK13, HLADQB1, 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 7.45 2.21E-01 HLA-DMA,HLA-DOA,HLA-DQA1,HLALymphocyte-mediated DRB1,HLA-DMB,HLA-DQB1,HLA-DPA1,HLAApoptosis of Target F,HLA-A,HLA-E,HLA-DRA,FCER1G,HLA-B,HLACells DOB,HLA-DQA2,HLA-G,HLA-DPB1,HLADRB5,HLA-C Crosstalk between 5.26 1.77E-01 NCR3, HLA-DRB1, LTB, CD83, TREM2, HLA-F, Dendritic Cells and MICB, HLA-A, HLA-E, LTA, HLA-DRA, HLA-B, Natural Killer Cells HLA-G, TNF, MICA, HLA-DRB5, HLA-C Altered T Cell and B 4.97 1.74E-01 HLA-DMA, HLA-DOA, SLAMF1, IL10, HLACell Signaling in DQA1, HLA-DRB1, LTB, HLA-DMB, HLA-DQB1, Rheumatoid Arthritis IL17A, LTA, HLA-DRA, FCER1G, HLA-DOB, TNF, HLA-DRB5 *The –log(p-value) significance threshold for this dataset is 1.2  128  Table A.6  Top significant gene association networks disrupted in RB1+/+ tumors.  ID 1  Molecules in Network 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  Score 50  Molecules 27  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  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 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 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  25  16  Energy Production, Lipid Metabolism, Small Molecule Biochemistry  23  15  Gene Expression, Skeletal and Muscular System Development and Function, Cellular Development  23  15  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/miR200b/miR-200c, MKI67, NR3C1, NXN, PDCD10, PPARA, PPP2CA, Sfk, SIKE1, SRC, TNF, TRAF3IP3, ZP2 AQP5, ARRB1, BYSL, CD34, CDH1, CDH11, CTNNB1, FCRL6, HLA-DRA, HLA-DRB4, HLADRB5, 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  23  15  Embryonic Development, Organismal Development, Reproductive System Development and Function Lipid Metabolism, Molecular Transport, Small Molecule Biochemistry  22  15  3  4  5  6  7  129  Top Functions Skeletal and Muscular System Development and Function, Tissue Morphology, Molecular Transport Cellular Movement, Cancer, Tissue Development  Cellular Movement, Cellular Development, Hair and Skin Development and Function  ID 8  Molecules in Network 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  Score 20  Molecules 14  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 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  20  14  19  13  10  130  Top Functions Cellular Development, Nervous System Development and Function, Cellular Assembly and Organization Amino Acid Metabolism, Carbohydrate Metabolism, Lipid Metabolism  Cardiovascular Disease, Cell Cycle, Cell Morphology  Table A.7 ID  Top significant gene association networks disrupted in RB1-/- tumors. 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  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  RNA PostTranscriptional Modification, Drug Metabolism, Endocrine System Development and Function 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 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 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  42  31  42  31  41  32  Endocrine System Development and Function, Infectious Disease, Lipid Metabolism  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, Cellmediated Immune Response, Cellular  5  6  7  131  DNA Replication, Recombination, and Repair, Energy Production, Nucleic Acid Metabolism Gene Expression, Tissue Development, Cell Death  ID  8  9  10  Molecules in Network  Score  EG:309638), TAF1C, TAF4 (includes EG:100149942), TAF8 (includes EG:129685), TBP, TCHH, TH1L, TOR1AIP2, ZNF76 ADORA1, CD48, G protein alpha, G protein alphai, Gprotein 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 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 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  132  Molecules  Top Functions Development  36  28  Cell Signaling, Molecular Transport, Nucleic Acid Metabolism  33  29  32  27  Tissue Morphology, Cellular Assembly and Organization, DNA Replication, Recombination, and Repair Cellular Assembly and Organization, Cellular Compromise, PostTranslational Modification  

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