Supplementary data Exome sequencing of hepatoblastoma reveals novel mutations and cancer genes in the Wnt pathway and ubiquitin ligase complex Deshui Jia 1,*, Rui Dong 2,*, Ying Jing 1, 3,*, Dan Xu 1,*, Qifeng Wang 4, Lei Chen 5, Qigen Li 6, Yuping Huang 1, Yuannv Zhang 1, Zhenfeng Zhang 1, Li Liu 1, Shan Zheng 2, Qiang Xia 6, Hongyang Wang 5,, Kuiran Dong 2,, Xianghuo He 1, To whom correspondence should be addressed:xhhe@shsci.org; kuirand@fudan.edu.cn; hywangk@vip.sina.com. This WORD file includes: Supplementary Materials and Methods Supplementary Figures S1 through S5 Other Supplementary Information for this manuscript includes the following: Supplementary Tables S1 through S9 1
Materials and Methods Ethics Statement Hepatoblastoma primary tumors and matched peripheral blood lymphocytes and adjacent non-tumor tissues were obtained from affected individuals undergoing surgical resection at the Children s Hospital of Fudan University, the Second Military Medical University and the Renji Hospital Affiliated to Shanghai Jiao Tong University School of Medicine. Participants that these samples were obtained from provided their written informed consent to participate in the study, and the Ethical Review Committee of the WHO Collaborating Center for Research in Human Production authorized by the Shanghai Municipal Government approved this study as well as the consent procedure. Sample selection and preparation for sequencing analyses DNA from 6 primary hepatoblastoma tumors and matched peripheral blood lymphocytes were obtained from affected individuals undergoing surgical resection at the Children s Hospital of Fudan University (Table 1 and Supplementary Table S1). None of the patients had received chemotherapy prior to resection. The tumor tissues were immediately frozen for short-term storage at -80 C. RNA was isolated using TRIzol reagent (Invitrogen) and DNA was isolated using the Qiagen reagents. Genomic DNA from tumor samples weighing up to 10 mg was extracted using the QIAamp DNA Micro Kit (Qiagen 56304) following the manufacturer s instructions. 2
Blood lymphocytes were derived from the same patient as the tumor DNA, separated and frozen for short-term storage at -80 C. Lymphocyte DNA was isolated using the QIAamp DNA Mini Kit (Qiagen 51304) and utilized as the normal germline control for genomic analyses. Illumina library construction and Illumina sequencing All methods for the library construction and whole-exome sequencing have been described previously 1. Genomic libraries were prepared using the Illumina TruSeq Sample DNA sample Preparation kit following the manufacturer s instructions. Enrichment was performed as previously described using the Illumina TruSeq Enrichment 62 Mb kit following the manufacturer s recommended protocol. Each exome was sequenced using the 100-bp paired-end protocol on an Illumina HiSeq 2000 DNA Analyzer to produce approximately 10-12 Gb of sequence per exome. The average exome sequencing coverage was 87.91% at 20 or above. Sequencing reads were aligned to the human reference genome (NCBI Build 37) using the Burrows-Wheeler Aligner (BWA) algorithm with the default setting. Reads that were unmapped, PCR-derived duplicates and reads mapping outside the targeted region of the genome were excluded from the analysis. The genotype files from Affymetrix SNP 6.0 microarrays were used for coverage analysis and for QC purposes. Identification and validation of somatic mutations A mismatched base was identified as a somatic mutation only when it was not present 3
in the matched normal sample. All putative somatic mutations were validated visually using the Integrated Genomics Viewer (IGV) and predicted to affect protein function by Polyphen (http://genetics.bwh.harvard.edu/pph). All non-synonymous mutations that were identified in the tumor samples were confirmed by independent PCR and sequencing in each tumor and its normal counterpart 2. Validated non-synonymous sequence variations were displayed using the Circos program 3. The 24 identified mutations within 21 somatically altered genes were further sequenced in a validation set composed of an independent series of 24 additional HB tumors and matched controls obtained from three hospitals, including the Children s Hospital of Fudan University (12 cases), the Second Military Medical University (8 cases) and the Renji Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (4 cases) (Supplementary Table S1). PCR amplification and Sanger sequencing analyses were performed following previously described protocols 2. SPOPmutations have previously been detected in various malignancies, and SPOP has clear mutational hotspots at specific codons in the region encoding the MATH domain 4-6. Therefore, we sequenced all SPOP exons in the validation set. However, resequencing all SPOP coding exons failed to identify any additional tumors with a somatic mutation in this gene. This result suggests that a somatic mutation in the SPOP gene is rare in HB tumors, consistent with previously reported results for breast, lung and liver cancer 7. In addition, we examined mutation status of the 24 identified mutations in two HB cell lines (HepG2 and HUH-6) and did not find any of those novel mutations in the two cell lines, except for the known β-catenin gene mutations 4
identified previously in HepG2 and HUH-6 8. SNP array analysis and data process The experiment was performed as previously described 1, 9. Primary HB tumors and paired lymphocyte samples were obtained from 6 HB patients (Supplementary Table S1). DNA was extracted, amplified and hybridized onto an Affymetrix Genome-Wide Human SNP array 6.0 according to the manufacturer s instructions (Affymetrix Inc.). The overall hybridization quality was estimated by the call rate index obtained from Genotyping Console Software (GTC 3.0, birdseed algorithm using default parameter settings). Partek Genomics Suite version 6.4 (Partek Inc., St. Louis, MO) was used for copy number analysis. Regions of CNAs were detected using a Hidden Markov Model (HMM) algorithm in the standard Partek workflow for paired samples. The following criteria were used to define significant CNAs were as follows: a gain or loss was inferred for a copy number of greater than 2.5 or less than 1.5, respectively; and the minimum physical length of the putative CNA was more than 100 kb. Gene annotation and overlap were conducted using the National Center for Biotechnology Information build 36.1 and the University of California, Santa Cruz (UCSC) hg18. Significant focal regions of amplification and deletion were identified by applying GISTIC algorithm with a threshold of 3.6/1.2 (log2 ratio of 0.848/20.737) as described previously 10. Focal regions of copy number alterations were displayed using the Circos program 3. 5
Gene expression analysis Total RNA was prepared from the HepG2 cells overexpressing vector control, SPOP wide-type and S119N mutant using TRIzol reagent (Invitrogen). The RNA was then labeled and hybridized to Affymetrix Human Genome U133 Plus 2.0 arrays following the manufacturer s instructions. Differential expression profiling was performed using an RVM (Random Variance Model) algorithm comparing the SPOP wide-type or mutant with vector control to identify differentially expressed genes (DEGs) (fold change 2). Gene Ontology and pathway enrichment analysis Pathway enrichment analyses of genes harboring somatic SNVs or CNA-related genes were performed using molecule annotation system (MAS), a web-based software toolkit (http://bioinfo.capitalbio.com/mas3/). Gene Ontology and pathway enrichment analyses were employed to determine the significant functions and pathways of the genes. Fisher s exact test was utilized to select the significant pathways and the threshold of significance was defined as a p-value<0.05 and q-value<0.001. Cell lines and cell culture Two HB cell lines (HepG2 and HUH-6) and HEK293T cells were cultured at 37 C in a 5% CO 2 atmosphere in DMEM medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. The cells were regularly certified as being free of mycoplasma contamination. 6
Lentiviral vector preparation and infection The lentiviral shrna vectors and the scrambled control shrna were purchased from Open Biosystems (Huntsville, AL, USA). Details of the shrna information are provided in the Supplementary Table 8. The lentiviral constructs for the SPOP, CTNNB1 and CAPRIN2 genes were constructed as described previously 9. The entire coding sequence of the target cdnas was amplified and cloned into the pwpxl vector, which was obtained from Addgene. The SPOP (S119N), CTNNB1 (S33F/G34V and G512V) and CAPRIN2 (R968H/S969C) mutants were generated using a Site-Directed Mutagenesis Kit (Stratagene). Details of the primer sequences and restriction sites are provided in the Supplementary Table 9. Lentivirus production was performed by cotransfecting the viral plasmids with 2 packaging plasmids with Lipofectamine 2000 into HEK293T cells. Supernatants containing the lentiviral particles were harvested at 48 h post-transfection and filtered. Infections were performed with viral supernatant in the presence of 8 μg/ml polybrene (Sigma-Aldrich). Quantitative PCR mrna expression levels were quantified using a 7500 Real-Time PCR System with SDS 2.3 software (Applied Biosystems) according to the manufacturer's instructions. Total RNA was extracted from 33 paired HB primary tumor and adjacent non-tumor tissues, which were reversely transcribed to complementary DNA (cdna) and then 7
subjected to q-pcr analysis of the mrna expression of candidate genes. First-strand cdna synthesis and amplification were performed using Reverse Transcription Reagents (Takara) according to the manufacturer's instructions. cdna templates were combined with SYBR Green premix containing Rox (Takara) to perform quantitative PCR reactions. β-actin was used as an endogenous control for mrna level optimization. Details of the primers sequences are provided in the Supplementary Table 9. The 33 paired HB primary tumor and adjacent non-tumor were obtained from three hospitals (Supplementary Table S1), including the Children s Hospital of Fudan University (16 cases), the Second Military Medical University (15 cases) and the Renji Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (2 cases). Cell proliferation assays Cells were seeded at 2000 cells/well in 96-well plates and incubated. An aliquot of 10 μl of Cell-Counting Kit (CCK)-8 (Dojindo, Kumamoto, Japan) was added to triplicate wells and incubated for 2 h. Subsequently, the absorbance was measured at 450 nm to calculate the number of viable cells in each well. Each measurement was performed in triplicate and the experiments were repeated twice. Colony formation assays Cells were seeded in 6-well plates at a concentration of 500 cells per well and cultured at 37 C for 2 wk. At the end of the incubation, the cells were fixed with 8
100% methanol and stained with 0.1% crystal violet. Megascopic cell colonies were counted by Image-Pro Plus 5.0 (Media Cybernetics, Bethesda, MD). Each measurement was performed in triplicate and each experiment was performed at least three times. Immunohistochemistry The experiments were performed as previously described 11. Immunohistochemistry (IHC) was performed on 5-mm sections of formalin-fixed paraffin-embedded (FFPE) tissue from 42 HB individuals (obtained from the surgical specimen archives of the Children s Hospital of Fudan University) by heat-induced epitope retrieval in EDTA (ph 9.0) using a pressure cooker treatment for 2 min. Samples were incubated at room temperature for 1 h with an antibodies against β-catenin (1:100; BD Biosciences), SPOP (1:600; Novus) and Caprin2 (1:00; Novus) followed by incubation with the secondary antibody (Dako); sections were counterstained with hematoxylin. Positive staining was defined according to the percentage of stained cells (0 = no cells, 1 = 1%-4%, 2 = 5%-19%, 3 = 20%-39%, 4 = 40%-59%, 5 = 60%-79%, 6 = 80%-100% of positive cells) and intensity (0-3). Percentage scores were multiplied by intensity scores to yield an overall score. A cytoplasmic and nuclear β-catenin score of >3 were considered as positive, whereas score of 0 to 3 was considered as negative. Cell cycle analysis Cells were fixed into 70% ethanol at 20 for 24 hours, stained with 50 μg/ml 9
propidium iodide (Kaiji, NanJing, China), and analyzed using Cytomics FC 500 MPL System (Beckman Coulter, CA). The results were analyzed using CXP software (Beckman Coulter, CA). Reporter Assay The experiments were performed as previously reported 12. Briefly, HUH-6 cells overexpressing GFP, CAPRIN2 WT and mutant constructs were seeded as triplicates in 24-well plates, respectively. Cells were transiently transfected with TOPFLASH or FOPFLASH plasmids (Millipore, Billerica, MA) with Renilla luciferase control vector (Promega, Madison, MI) using Lipofectamine 3000 (Invitrogen). Luciferase activity was measured with a dual-luciferase kit (Promega). Co-immunoprecipitation (Co-IP) Co-IP experiments were performed with the Pierce Co-Immunoprecipitation (Co-IP) Kit (Thermo Scientific) according to the manufacturer's instructions. Whole cell lysates were collected from the specified cells and subjected to co-ip analysis. The antibody against Androgen receptor (Santa Cruz Biotechnology) was used for co-ip. Nuclear and cytoplasmic protein extraction Nuclear and cytoplasmic protein extractions were performed by following the manufacturer s instructions of NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific). HepG2 and HUH6 cells overexpressing GFP, CAPRIN2 WT and 10
mutant constructs were used to extract nuclear and cytoplasmic proteins. Specific antibodies against α-tubulin and Lamin A/C were used as cytoplasmic and nuclear protein control. Immunoblotting analysis For immunoblotting analysis, cells were lysed and protein concentration was determined by the Bradford assay. The cell lysates were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blocked and incubated with specific primary antibodies against SPOP (Thermo Scientific), β-catenin (BD), α-tubulin (Cell Signaling Technology), Lamin A/C (Cell Signaling Technology), Androgen receptor (Cell Signaling Technology), Caprin2 (Proteintech), CDKN2B (Abcam), Ubiquitin (Abcam) or β-actin (Sigma). Statistical analysis Statistical analysis and graphical presentation were performed with GraphPad Prism 5.0. The results are presented as the mean ± s.e.m and were evaluated using an unpaired Student s t-test (two-tailed; p<0.05 was considered to be significant), unless specified otherwise (paired t-test and non-parametric test). Statistical analysis of differences between study groups was performed using an unpaired Student s t-test and p<0.05 was considered statistically significant. A paired Student s t-test was used to analyze differences in the expression of mrna levels among tumors and paired non-tumor tissues in q-pcr analysis. 11
Supplementary Figures Supplementary Figure S1. The genomic landscape of somatic copy number alterations in 6 HB samples. Chromosomes are depicted one by one. Samples (HB2, HB3, HB5, HB6, HB8 and HB9) are represented in each chromosome. Amplifications are shown in blue, while deletions are shown in red. 12
Supplementary Figure S2. shrna-mediated loss-of-function screening of mutant genes on HB cell proliferation. A. CCK-8 assays depicting representative cell proliferation results in HepG2 and HUH-6 cells after lentivirus-mediated knockdown of OR5I1 and CDC20B genes. B. Colony formation assay showing the effects of knockdown of the SPOP, OR5I1 and CDC20B genes on the proliferation of HepG2 and HUH-6 cells. C. Colony formation assay showing the effects of knockdown of the CTNNB1 and CAPRIN2 genes on the proliferation of HepG2 and HUH-6 cells. D. 13
Representative results of CCK-8 assays for the effects of knockdown of KLHL22, GLTSCR1 and TMC5 genes on the in vitro proliferation of HepG2 cells by lentivirus-mediated knockdown. E. Representative results of CCK-8 assays for the effects of knockdown of VCAM1, TRPC4AP and FGGY genes on the in vitro proliferation of HUH-6 cells by lentivirus-mediated knockdown. The results are shown as mean ± s.e.m, *, p<0.05; **, p<0.01; ***, p<0.001. 14
Supplementary Figure S3. q-pcr analyses of mrna levels of mutant genes in 33 paired HB and adjacent non-tumor tissues. q-pcr analyses of the expression levels of 6 mutant genes (VCAM1, RNF169, HDCA9, FGGY, C2orf57, GLTSCR1, CAPRIN2, TRPC4AP and KLHL22). p< 0.05 was considered significant. 15
Supplementary Figure S4. SPOP S119N is a loss-of-function mutation in HB. A. Representative images of colony formation assays showing GFP, wild-type (WT) SPOP or SPOP S119N overexpression in HepG2 and HUH-6 cells. B. Representative results of cell cycle analyses by FACS. Knockdown of the SPOP promotes the cell cycle progression from G1 to S phase in HepG2 cells. C. q-pcr analyses of the knockdown of SPOP gene on the expression levels of CDKN2B in HepG2 and HUH-6 cells, respectively. 16
Supplementary Figure S5. SPOP promotes ubiquitination of Androgen receptor. Overexpression of SPOP increases ubiquitination of endogenous AR, whereas overexpression of SPOP S119N mutant inhibits the ubiquitination of AR. HepG2 cells empty vector (HepG2-GFP), SPOP WT (HepG2-SPOP WT) and SPOP S119N (HepG2-SPOP S119N) were treated with 10 mm MG132 for 8 hr. Immunoprecipitated AR proteins were analyzed by WB for ubiquitination. 17
Supplementary Tables Supplementary Table S1. Numbers of HB tumors analyzed by each platform Supplementary Table S2. Somatic sequence variations identified in 6 HB genomes by whole-exome sequencing. Supplementary Table S3. Valdiation of 21 identified mutations in an additional set of 24 HB tumors by sanger sequencing. Supplementary Table S4. Somatic copy number alterations identified in 6 HB genomes by SNP 6.0 arrays. Supplementary Table S5. Focal regions of amplifications and deletions identified in 6 HB genomes. Supplementary Table S6. Immunohistochemical analysis of SPOP and Caprin2 proteins in HB tumors. Supplementary Table S7. The identification of differentially expressed genes in HepG2 cells overexpressing empty vector, SPOP WT and S119N mutant by microarray. In order to identify regulators that mediate SPOP-induced cell cycle arrest, we employed gene expression microarray to explore candidate genes among HepG2 cells overexpressing empty vector (GFP), SPOP wide type (WT) and SPOP S119N mutant. The HepG2-GFP cells were used as reference. Upregulation, fold change 2 and downregulation, fold change 0.5. Supplementary Table S8. The sequences of sirnas and shrna plasmids used. Supplementary Table S9. Primers used for q-pcr and subclone. 18
References 1. Barbieri CE, Baca SC, Lawrence MS, Demichelis F, Blattner M, Theurillat JP, White TA, et al. Exome sequencing identifies recurrent SPOP, FOXA1 and MED12 mutations in prostate cancer. Nat Genet 2012;44:685-689. 2. Huang J, Deng Q, Wang Q, Li KY, Dai JH, Li N, Zhu ZD, et al. Exome sequencing of hepatitis B virus-associated hepatocellular carcinoma. Nat Genet 2012;44:1117-1121. 3. Krzywinski M, Schein J, Birol I, Connors J, Gascoyne R, Horsman D, Jones SJ, et al. Circos: an information aesthetic for comparative genomics. Genome Res 2009;19:1639-1645. 4. Geng C, He B, Xu L, Barbieri CE, Eedunuri VK, Chew SA, Zimmermann M, et al. Prostate cancer-associated mutations in speckle-type POZ protein (SPOP) regulate steroid receptor coactivator 3 protein turnover. Proc Natl Acad Sci U S A 2013;110:6997-7002. 5. Kim MS, Je EM, Oh JE, Yoo NJ, Lee SH. Mutational and expressional analyses of SPOP, a candidate tumor suppressor gene, in prostate, gastric and colorectal cancers. APMIS 2013;121:626-633. 6. Le Gallo M, O'Hara AJ, Rudd ML, Urick ME, Hansen NF, O'Neil NJ, Price JC, et al. Exome sequencing of serous endometrial tumors identifies recurrent somatic mutations in chromatin-remodeling and ubiquitin ligase complex genes. Nat Genet 2012;44:1310-1315. 7. Kim MS, Yoo NJ, Lee SH. Somatic mutation of SPOP tumor suppressor gene is rare in breast, lung, liver cancers, and acute leukemias. APMIS 2013. 8. Cairo S, Armengol C, De Reynies A, Wei Y, Thomas E, Renard CA, Goga A, et al. Hepatic stem-like phenotype and interplay of Wnt/beta-catenin and Myc signaling in aggressive childhood liver cancer. Cancer Cell 2008;14:471-484. 9. Jia D, Wei L, Guo W, Zha R, Bao M, Chen Z, Zhao Y, et al. Genome-wide copy number analyses identified novel cancer genes in hepatocellular carcinoma. Hepatology 2011;54:1227-1236. 10. Weir BA, Woo MS, Getz G, Perner S, Ding L, Beroukhim R, Lin WM, et al. Characterizing the cancer genome in lung adenocarcinoma. Nature 2007;450:893-898. 11. Lachenmayer A, Alsinet C, Savic R, Cabellos L, Toffanin S, Hoshida Y, Villanueva A, et al. Wnt-pathway activation in two molecular classes of hepatocellular carcinoma and experimental modulation by sorafenib. Clin Cancer Res 2012;18:4997-5007. 12. Li J, Zhou BP. Activation of beta-catenin and Akt pathways by Twist are critical for the maintenance of EMT associated cancer stem cell-like characters. BMC Cancer 2011;11:49. 19
Supplementary Table S1 Numbers of HB tumors analyzed by each platform Data type Primary tumors Lymphocytes adjacent Exome sequencing 6 6 0 SNP 6.0 array 6 6 0 Expression array a 5 0 5 Exome sequencing, SNP 6.0 array b 6 6 0 Exome sequencing, SNP 6.0 and Expression array c 3 3 3 PCR + Sanger Sequencing d 24 6 18 quantitative-pcr (q-pcr) e 33 0 33 Immunohistochemistry (IHC) f 42 0 10 a, Expression arrays were used to analyze the 5 paired HB primary tumors and adjacent non-tumor liver tissues. b, Exome livers sequencing and SNP 6.0 array were used to analyze the same 6 paired HB primary tumors and lymphocytes. c, 3 HB primary tumors were analyzed by Exome sequencing, SNP 6.0 array and Expression arrays. d, PCR amplification and Sanger sequencing were examined in an additional set of 24 paired HB primary tumors and lymphocytes or adjacent non-tumor tissues. e, q-pcr was employed to examine the relative expression levels of 21 mutant genes in 33 paired HB primary tumors and adjacent non-tumor tissues. f, IHC was employed to examine the protein expression levels of β-catenin in an overlapping or additional set of 42 HB primary tumors and 10 non-tumor tissues.
Supplementary Table S2. Somatic sequence variations identified in 6 HB genomes by whole-exome sequencing. Chromosome Start End REF ALT QUAL Samples Gene_location Exon_syno Gene chr1 34663092 34663092 G C 717.04 HB2 exonic nonsynonymous SNV C1orf94 chr2 33173826 33173826 C T 273.49 HB2 intronic NA LTBP1 chr2 208994697 208994697 C T 788 HB2 ncrna_intronic NA LOC100507443 chr2 209000144 209000144 G A 1408.7 HB2 ncrna_intronic NA LOC100507443 chr2 212251864 212251864 T C 1988.47 HB2 exonic synonymous SNV ERBB4 chr2 216182770 216182770 C T 1319.66 HB2 intronic NA ATIC chr2 216191533 216191533 C T 1378.02 HB2 intronic NA ATIC chr2 216982370 216982370 C G 6472.46 HB2 intronic NA XRCC5 chr2 218712936 218712936 C T 1533.2 HB2 exonic synonymous SNV TNS1 chr2 219210781 219210781 G A 2484.39 HB2 UTR3 NA PNKD chr2 219232354 219232354 C A 862.2 HB2 intronic NA C2orf62 chr2 220251208 220251208 T C 2511.93 HB2 intronic NA DNPEP chr2 226491645 226491645 A T 607.61 HB2 exonic nonsynonymous SNV NYAP2 chr2 226517389 226517389 C A 2355.44 HB2 UTR3 NA NYAP2 chr2 227966148 227966148 C T 2011.26 HB2 intronic NA COL4A4 chr2 228177567 228177567 C T 3073.53 HB2 UTR3 NA COL4A3 chr2 228221725 228221725 A G 3443.37 HB2 exonic synonymous SNV MFF chr2 228227060 228227060 C A 1307.34 HB2 UTR3 NA TM4SF20 chr2 228734956 228734956 G A 1962.18 HB2 intergenic NA CCL20(dist=52676),WDR69(dist=1371) chr2 228750239 228750239 G T 1521.97 HB2 intronic NA WDR69 chr2 228996650 228996650 A G 6286.39 HB2 intronic NA SPHKAP chr2 231226488 231226488 C T 1383.5 HB2 intronic NA SP140L chr2 232458818 232458818 C T 2115.34 HB2 exonic nonsynonymous SNV C2orf57 chr2 233282847 233282847 C G 4175.9 HB2 intergenic NA ALPPL2(dist=7423),ALPI(dist=37986)
chr2 234825895 234825895 C G 2595.73 HB2 upstream NA TRPM8 chr2 234873503 234873503 G A 1554.03 HB2 intronic NA TRPM8 chr2 242028282 242028282 C T 2752.13 HB2 intronic NA SNED1 chr2 242032540 242032540 C A 1556.34 HB2 UTR3 NA SNED1 chr2 242047750 242047750 A G 3140.74 HB2 intronic NA PASK chr2 242058214 242058214 A G 952.48 HB2 intronic NA PASK chr2 242077299 242077299 G A 2055.5 HB2 intronic NA PASK chr2 242082218 242082218 C T 1128.72 HB2 intronic NA PASK chr2 242122037 242122037 G C 4622.38 HB2 intronic NA PPP1R7 chr3 38404513 38404513 G A 846.1 HB2 intronic NA XYLB chr3 41266104 41266104 G T 1093.09 HB2 exonic nonsynonymous SNV CTNNB1 chr9 15623462 15623462 T C 1055.06 HB2 intronic NA CCDC171 chr9 43151833 43151833 G A 2424.23 HB2 intergenic NA LOC642929(dist=6349),FAM75A6(dist=472669) chr10 133976706 133976706 G T 2048.02 HB2 intronic NA JAKMIP3 chr11 74547510 74547510 G T 160.49 HB2 exonic nonsynonymous SNV RNF169 chr17 48699116 48699116 T C 4611.32 HB2 exonic synonymous SNV CACNA1G chr18 77929368 77929368 A C 573.97 HB2 ncrna_exonic NA LOC100130522 chr19 16345923 16345923 T A 623.38 HB2 UTR3 NA AP1M1 chr19 16345924 16345924 G C 702.8 HB2 UTR3 NA AP1M1 chr19 16345925 16345925 T A 743.36 HB2 UTR3 NA AP1M1 chr19 48198194 48198194 G A 663.03 HB2 exonic nonsynonymous SNV GLTSCR1 chr22 20843498 20843498 T A 357.72 HB2 exonic nonsynonymous SNV KLHL22 chr22 20843499 20843499 C T 393.35 HB2 UTR5 NA KLHL22 chrx 48933180 48933180 C T 1836.34 HB2 intronic NA WDR45 chrx 52891738 52891738 C T 76.56 HB2 intronic NA XAGE3 chrx 52891739 52891739 A G 88.86 HB2 intronic NA XAGE3
chr1 17046549 17046549 G A 2303 HB3 ncrna_exonic NA ESPNP chr1 101196970 101196970 T C 500.22 HB3 exonic nonsynonymous SNV VCAM1 chr1 142671117 142671117 C T 94.67 HB3 intergenic NA NONE(dist=NONE),LOC375010(dist=26304) chr1 142671150 142671150 G A 99.91 HB3 intergenic NA NONE(dist=NONE),LOC375010(dist=26271) chr2 91795381 91795381 A C 1575.15 HB3 intergenic NA NONE(dist=NONE),LOC654342(dist=29328) chr3 49739276 49739276 C T 606.74 HB3 exonic synonymous SNV RNF123 chr5 54423155 54423155 G A 744.36 HB3 exonic nonsynonymous SNV CDC20B chr5 176492778 176492778 C T 63.37 HB3 UTR3 NA ZNF346 chr6 7883226 7883226 T G 274.09 HB3 ncrna_exonic NA MUTED-TXNDC5 chr6 152793575 152793575 A G 3154.86 HB3 intronic NA SYNE1 chr7 7557296 7557296 C A 52.78 HB3 intronic NA COL28A1 chr7 8095016 8095016 G A 545.03 HB3 intronic NA GLCCI1 chr7 20666060 20666060 C A 93.3 HB3 intronic NA ABCB5 chr7 107356693 107356693 T C 264.34 HB3 UTR3 NA SLC26A4 chr11 79698784 79698784 C A 406.18 HB3 intergenic NA ODZ4(dist=547089),MIR4300(dist=1902999) chr17 47696467 47696467 C T 349.72 HB3 exonic nonsynonymous SNV SPOP chr17 80573272 80573272 A G 55.39 HB3 UTR3 NA WDR45L chr20 44685193 44685193 A T 347.95 HB3 exonic nonsynonymous SNV SLC12A5 chry 13266301 13266301 A G 1786.07 HB3 intergenic NA NONE(dist=NONE),GYG2P1(dist=1251614) chrun_gl000229 16677 16677 G A 114.39 HB3 intergenic NA NONE(dist=NONE),NONE(dist=NONE) chr5 167988433 167988433 T A 67.77 HB5 exonic nonsynonymous SNV PANK3 chr6 10756728 10756728 C T 104.16 HB5 exonic nonsynonymous SNV TMEM14B chr9 88990407 88990407 C T 174.13 HB5 intergenic NA ZCCHC6(dist=21005),GAS1(dist=568870) chr9 88990413 88990413 C T 174.05 HB5 intergenic NA ZCCHC6(dist=21011),GAS1(dist=568864) chr10 102732382 102732382 T A 221.86 HB5 UTR5 NA SEMA4G chr14 100363672 100363672 A G 1888.09 HB5 intronic NA EML1
chr17 27083757 27083757 C T 56.73 HB5 UTR3 NA C17orf63 chrun_gl000214 31265 31265 C A 122.33 HB5 intergenic NA NONE(dist=NONE),NONE(dist=NONE) chrun_gl000220 134139 134139 G A 76.1 HB5 intergenic NA LOC100507412(dist=7443),RN5-8S1(dist=21858) chrun_gl000220 134143 134143 A G 79.99 HB5 intergenic NA LOC100507412(dist=7447),RN5-8S1(dist=21854) chr1 63307298 63307298 A T 55.59 HB6 intronic NA ATG4C chr1 152975715 152975715 C T 133.22 HB6 exonic synonymous SNV SPRR3 chr1 245021253 245021253 C A 50.31 HB6 intronic NA HNRNPU chr3 41266101 41266101 C T 666.92 HB6 exonic nonsynonymous SNV CTNNB1 chr3 41275640 41275640 G T 1014.02 HB6 exonic nonsynonymous SNV CTNNB1 chr5 64511377 64511377 T G 85.09 HB6 intronic NA ADAMTS6 chr6 132195555 132195555 C A 54.52 HB6 intronic NA ENPP1 chr8 100169030 100169030 A T 56.32 HB6 intronic NA VPS13B chr8 100169032 100169032 G A 53.43 HB6 intronic NA VPS13B chr9 103275692 103275692 T A 1323.53 HB6 intronic NA MSANTD3-TMEFF1,TMEFF1 chr10 101088828 101088828 C T 151.75 HB6 upstream NA CNNM1 chr11 10522983 10522983 G T 864.47 HB6 intronic NA AMPD3 chr11 55703713 55703713 G A 334.36 HB6 exonic nonsynonymous SNV OR5I1 chr15 36984413 36984413 C T 496.49 HB6 intronic NA C15orf41 chr16 19498525 19498525 T C 328.17 HB6 exonic nonsynonymous SNV TMC5 chrx 76918844 76918844 T C 53.71 HB6 intronic NA ATRX chrx 154344314 154344314 G A 525 HB6 intronic NA BRCC3 chr1 53580601 53580601 C T 558.01 HB8 exonic nonsynonymous SNV SLC1A7 chr1 106240709 106240709 C T 1640.09 HB8 intergenic NA LOC100129138(dist=1621016),PRMT6(dist=1358558) chr1 179494643 179494643 T C 121.13 HB8 intronic NA AXDND1 chr1 186322862 186322862 A T 617.26 HB8 exonic synonymous SNV TPR chr1 200521186 200521186 C T 1144.1 HB8 UTR3 NA KIF14
chr1 207991723 207991723 C T 191.94 HB8 upstream NA LOC148696 chr3 169712432 169712432 T C 759.54 HB8 UTR3 NA SEC62 chr3 183529179 183529179 T C 172.62 HB8 UTR3 NA YEATS2 chr5 36984945 36984945 G A 2900.22 HB8 exonic nonsynonymous SNV NIPBL chr7 18993868 18993868 T A 352.57 HB8 exonic nonsynonymous SNV HDAC9 chr8 69552682 69552682 C T 957.43 HB8 exonic nonsynonymous SNV C8orf34 chr10 38369124 38369124 C G 412.5 HB8 intergenic NA ZNF33A(dist=20129), ZNF37A(dist=14140) chr10 70056760 70056760 G A 2756.37 HB8 intronic NA PBLD chr11 869734 869734 C T 1538.57 HB8 UTR3 NA CHID1 chr12 7520715 7520715 C T 2424.26 HB8 exonic nonsynonymous SNV CD163L1 chr12 30863315 30863315 T A 702.98 HB8 exonic nonsynonymous SNV CAPRIN2 chr12 30863317 30863317 C T 723.08 HB8 exonic nonsynonymous SNV CAPRIN2 chr14 21762896 21762896 G A 74.51 HB8 exonic nonsynonymous SNV RPGRIP1 chr15 75311842 75311842 G C 2681.85 HB8 UTR3 NA SCAMP5 chr16 70433328 70433328 T A 183.57 HB8 UTR5 NA ST3GAL2 chr17 9818967 9818967 C T 1069.06 HB8 UTR3 NA GAS7 chr17 73774477 73774477 A T 1691.03 HB8 UTR3 NA H3F3B chr18 5237531 5237531 C T 631.47 HB8 ncrna_exonic NA LINC00526 chr18 14809923 14809923 A G 668.01 HB8 intronic NA ANKRD30B chr18 24432387 24432387 C T 476.94 HB8 UTR3 NA AQP4 chr21 40668979 40668979 G A 965.6 HB8 UTR3 NA BRWD1 chr22 23705420 23705420 C G 283.72 HB8 ncrna_intronic NA CES5AP1 chrx 52652326 52652326 A C 779.78 HB8 ncrna_intronic NA SSX8 chrx 53656390 53656390 C A 222.16 HB8 intronic NA HUWE1 chrx 76139673 76139673 T A 311.69 HB8 downstream NA MIR384 chrx 119512695 119512695 G C 846.78 HB8 intronic NA ATP1B4
chrx 149887056 149887056 C T 2502.61 HB8 intronic NA MTMR1 chrx 152957711 152957711 C A 2069.04 HB8 intronic NA SLC6A8 chrx 152957712 152957712 C A 2101.94 HB8 intronic NA SLC6A8 chr1 60073526 60073526 G T 1934.89 HB9 exonic nonsynonymous SNV FGGY chr1 145109456 145109456 C T 341.58 HB9 intronic NA SEC22B chr1 202287554 202287554 T C 70.79 HB9 exonic nonsynonymous SNV LGR6 chr1 220205704 220205704 T A 2363.41 HB9 ncrna_intronic NA RNU5F-1 chr4 1333727 1333727 C G 2316.71 HB9 UTR3 NA MAEA chr10 1205736 1205736 A G 119.48 HB9 ncrna_exonic NA LINC00200 chr15 50971685 50971685 A T 243.75 HB9 intronic NA TRPM7 chr15 50971686 50971686 T A 221.71 HB9 intronic NA TRPM7 chr18 33613595 33613595 T C 319.12 HB9 intronic NA RPRD1A chr18 47093790 47093790 C T 787.44 HB9 intronic NA LIPG chr20 33592368 33592368 C T 1219.66 HB9 exonic nonsynonymous SNV TRPC4AP chrx 23804197 23804197 A G 2323.4 HB9 UTR3 NA SAT1 chrx 30248973 30248973 G A 786.84 HB9 intronic NA MAGEB3
Supplementary Table S3 Valdiation of 21 identified mutations in an additional set of 24 HB tumors by sanger sequencing. Gene symbol Mutation prevalence Mutated residues CTNNB1 7/24 In-frame deletion (exon 3) OR5I1 2/24 In-frame deletion (exon 1) C2ORF57 1/24 R386C
Supplementary Table S4. Somatic copy number alterations identified in 6 HB genomes by SNP 6.0 arrays. Chromosome Cytoband Start End Length(bps) Copy number state Mean Sample ID chr2 2q32.3 194696957 195343992 647036 Amplification 4.02614 HB2 chr2 2q24.1 158553222 159213740 660519 Amplification 3.96242 HB2 chr2 2q24.2 159837141 160507273 670133 Amplification 3.97385 HB2 chr2 2q37.1 234466843 235241645 774803 Amplification 3.14959 HB2 chr2 2q32.3 193782168 194696957 914790 Amplification 2.73377 HB2 chr2 2q34 212423358 213477266 1053909 Amplification 2.62736 HB2 chr2 2q32.3 195343992 196455835 1111844 Amplification 2.72231 HB2 chr2 2q32.1-2q32.2 188923022 190284518 1361497 Amplification 2.73984 HB2 chr2 2q36.3-2q37.1 230344980 233775948 3430969 Amplification 2.80939 HB2 chr2 2q32.2-2q32.3 190284518 193782168 3497651 Amplification 4.29698 HB2 chr1 1q21.1-1q21.3 147187802 151652842 4465041 Amplification 3.27729 HB2 chr2 2q33.3-2q34 205638001 212423053 6785053 Amplification 2.79569 HB2 chr2 2q37.1-2q37.3 235241645 242293274 7051630 Amplification 2.74546 HB2 chr2 2q32.3-2q33.3 196455835 205638001 9182167 Amplification 4.32689 HB2 chr2 2q34-2q36.3 214876875 228919703 14042829 Amplification 2.80475 HB2 chr2 2q24.2-2q32.1 161678025 188923022 27244998 Amplification 3.82864 HB2 chr1 1q41-1q44 218003177 247191012 29187836 Amplification 3.30328 HB2 chr1 1q21.3-1q41 151654944 218002677 66347734 Amplification 3.29308 HB2 chr14 14q11.2 21553603 22045653 492051 Amplification 2.65561 HB3 chr1 1q21.1 144119772 146847073 2727302 Amplification 2.6162 HB3 chr14 14q11.2 21544692 22069312 524621 Amplification 2.64529 HB5 chr20 20q13.13 48579984 48787756 207773 Amplification 2.58937 HB6 chr14 14q11.2 21603762 22039983 436222 Amplification 2.92074 HB6 chr7 7q34 141734268 142211949 477682 Amplification 2.67248 HB6 chr9 9p11.2-9q12 44107100 69231871 25124772 Amplification 2.52906 HB6 chry Yp11.31 2710438 2811696 101259 Amplification 2.68248 HB8 chr17 17p13.3 2235262 2336957 101696 Amplification 2.93056 HB8 chr12 12q23.1 96529628 96632185 102558 Amplification 3.27158 HB8 chr17 17q25.3 78130070 78232835 102766 Amplification 3.10014 HB8 chr1 1q41 212135144 212238934 103791 Amplification 2.70702 HB8 chr10 10q11.22 46374535 46479659 105125 Amplification 2.93436 HB8 chr6 6p12.1 52740417 52846250 105834 Amplification 2.75623 HB8 chr12 12q15 68142653 68250974 108322 Amplification 3.31593 HB8 chr13 13q21.2-13q21.31 60393315 60502038 108724 Amplification 2.52762 HB8 chr2 2q21.2-2q21.3 134763876 134873064 109189 Amplification 2.66999 HB8 chr1 1q31.2 191907505 192017264 109760 Amplification 2.58187 HB8 chr2 2q37.3 242628107 242738130 110024 Amplification 2.80427 HB8 chr1 1q44 242481787 242591953 110167 Amplification 3.3524 HB8 chr17 17p11.2 19395068 19505574 110507 Amplification 2.60624 HB8 chr8 8q24.13 123848738 123960237 111500 Amplification 2.67057 HB8 chr8 8q24.3 144128966 144241387 112422 Amplification 2.50579 HB8 chr12 12q21.31 84608257 84721414 113158 Amplification 2.55452 HB8
chr10 10p15.1 5313280 5426726 113447 Amplification 2.58471 HB8 chr17 17q24.1 60442205 60555683 113479 Amplification 2.57644 HB8 chr12 12q21.1 70194846 70308440 113595 Amplification 2.78268 HB8 chr12 12q21.1 71840200 71955113 114914 Amplification 2.67065 HB8 chr12 12q23.2 102229740 102345270 115531 Amplification 2.92885 HB8 chr1 1q25.1 173518478 173634073 115596 Amplification 2.88218 HB8 chr12 12q24.23 117147476 117264592 117117 Amplification 2.62916 HB8 chr12 12q13.13 49258861 49376942 118082 Amplification 2.82733 HB8 chr1 1q31.3 193197429 193315771 118343 Amplification 2.54778 HB8 chr17 17p13.3 526 119081 118556 Amplification 2.55885 HB8 chr12 12p12.1 24350030 24468814 118785 Amplification 2.67453 HB8 chr6 6p24.3 7469496 7590756 121261 Amplification 2.74183 HB8 chr12 12q21.1 73029446 73153469 124024 Amplification 2.61157 HB8 chr20 20q13.13 47334164 47458307 124144 Amplification 2.50921 HB8 chr12 12q15 66542752 66667388 124637 Amplification 2.71751 HB8 chr12 12q23.3 104212164 104336925 124762 Amplification 2.51027 HB8 chr1 1q32.1 203264384 203389610 125227 Amplification 3.01015 HB8 chr19 19q12 33585497 33711085 125589 Amplification 2.59046 HB8 chr16 16q11.2-16q12.1 45388647 45515290 126644 Amplification 2.64732 HB8 chr8 8q24.13 124471533 124598387 126855 Amplification 2.5817 HB8 chr17 17q25.3 76880130 77007388 127259 Amplification 2.7852 HB8 chr6 6q13 74283003 74411086 128084 Amplification 2.76673 HB8 chr12 12p13.1 13575331 13704189 128859 Amplification 2.91441 HB8 chr1 1q21.3 152007488 152136863 129376 Amplification 2.67288 HB8 chr12 12q23.2 100091690 100221813 130124 Amplification 2.7735 HB8 chr1 1q42.2 231533826 231664241 130416 Amplification 2.84183 HB8 chr1 1q41 219032628 219163466 130839 Amplification 2.74459 HB8 chr1 1q24.3 169453950 169585299 131350 Amplification 2.69975 HB8 chr10 10p12.33 17381298 17512664 131367 Amplification 2.53714 HB8 chr2 2p13.1 75220505 75352367 131863 Amplification 2.56229 HB8 chr12 12q14.2 61662221 61794371 132151 Amplification 2.8011 HB8 chr12 12q12 43501256 43633570 132315 Amplification 3.20466 HB8 chr10 10p13 14672508 14805706 133199 Amplification 2.50442 HB8 chr10 10q21.1 57771396 57904944 133549 Amplification 2.5347 HB8 chry Yp11.2 7923944 8057743 133800 Amplification 2.7203 HB8 chr12 12q24.31 120013634 120148040 134407 Amplification 3.16297 HB8 chr12 12q21.1 70848830 70983472 134643 Amplification 2.51027 HB8 chr12 12q21.31 78948515 79083198 134684 Amplification 2.88608 HB8 chr12 12p12.3 17344066 17479835 135770 Amplification 2.59063 HB8 chr8 8q24.13 125612717 125749178 136462 Amplification 2.57016 HB8 chr1 1q25.1 173637024 173773842 136819 Amplification 2.78015 HB8 chr12 12q21.1 73469291 73606603 137313 Amplification 2.71926 HB8 chr1 1p36.32 3572469 3710855 138387 Amplification 2.70959 HB8 chr12 12p12.1 25128780 25267690 138911 Amplification 2.97038 HB8
chr12 12q24.32 125093787 125233195 139409 Amplification 2.75598 HB8 chr1 1q23.3 162740047 162880879 140833 Amplification 3.00495 HB8 chr20 20p12.2 11675944 11816853 140910 Amplification 2.50274 HB8 chr1 1q32.2 206902409 207044206 141798 Amplification 2.76418 HB8 chr1 1q21.2 148205108 148346926 141819 Amplification 3.08071 HB8 chr13 13q14.12 44317340 44459499 142160 Amplification 2.5114 HB8 chr2 2q23.3 150409156 150551755 142600 Amplification 2.61072 HB8 chr1 1q23.3 158982941 159125782 142842 Amplification 2.81442 HB8 chr12 12q21.1 70046647 70189577 142931 Amplification 2.98031 HB8 chr1 1q44 242335473 242478915 143443 Amplification 2.65698 HB8 chr12 12p11.21 32076550 32220663 144114 Amplification 3.156 HB8 chr12 12q24.31 119868882 120013166 144285 Amplification 3.01656 HB8 chr12 12q13.11-12q13.12 47337561 47482451 144891 Amplification 3.11242 HB8 chr12 12q21.31 79363561 79508930 145370 Amplification 2.80077 HB8 chr12 12q21.33 87822931 87968473 145543 Amplification 2.70211 HB8 chr12 12q24.31 120149895 120295666 145772 Amplification 3.66685 HB8 chr2 2q36.2 225184389 225330470 146082 Amplification 2.51187 HB8 chr1 1q32.1 203115451 203262644 147194 Amplification 2.97028 HB8 chr1 1q41 218393775 218541945 148171 Amplification 2.80429 HB8 chr12 12q24.21 113264872 113413642 148771 Amplification 2.97044 HB8 chr12 12p12.3 18273955 18423129 149175 Amplification 2.60266 HB8 chr1 1q31.3 193895942 194045967 150026 Amplification 2.65737 HB8 chr12 12q24.21 112842520 112992589 150070 Amplification 3.12578 HB8 chr12 12q23.2 100980066 101130525 150460 Amplification 2.99883 HB8 chr2 2q13 110178400 110329299 150900 Amplification 2.73134 HB8 chr8 8q11.21 49014144 49165315 151172 Amplification 2.56244 HB8 chr13 13q13.1 31802052 31953907 151856 Amplification 2.54553 HB8 chr8 8q24.13 126600330 126752502 152173 Amplification 2.57166 HB8 chr1 1q31.3 194048478 194200794 152317 Amplification 2.68478 HB8 chr6 6p21.31 35674670 35827566 152897 Amplification 2.51961 HB8 chr12 12p12.1 24975388 25128593 153206 Amplification 2.70667 HB8 chr1 1q43 238112084 238266368 154285 Amplification 2.83753 HB8 chr17 17p13.3 2472264 2626578 154315 Amplification 2.52677 HB8 chr1 1q25.3 182520506 182675094 154589 Amplification 2.91464 HB8 chr1 1q31.2 191749940 191907195 157256 Amplification 2.53817 HB8 chr12 12p12.1 25726129 25886139 160011 Amplification 2.68767 HB8 chr1 1q22 154552269 154713258 160990 Amplification 3.03872 HB8 chr2 2p24.3 13472933 13634088 161156 Amplification 2.73698 HB8 chr1 1q44 241932748 242094148 161401 Amplification 3.0358 HB8 chr1 1q32.1 200496665 200658640 161976 Amplification 2.85102 HB8 chr12 12q12 44355290 44517786 162497 Amplification 2.82408 HB8 chr2 2p11.2 89741711 89904417 162707 Amplification 2.53109 HB8 chr12 12p13.31 5427425 5590837 163413 Amplification 2.84744 HB8 chr6 6q12 64123193 64286722 163530 Amplification 2.58383 HB8
chr16 16q22.1 67744506 67908036 163531 Amplification 2.57941 HB8 chr1 1q25.3 181197863 181362077 164215 Amplification 2.99074 HB8 chr10 10q24.31 102059046 102223922 164877 Amplification 2.59539 HB8 chr17 17q21.32 44373235 44538606 165372 Amplification 2.82199 HB8 chr1 1q32.3 209541032 209706496 165465 Amplification 3.04103 HB8 chr12 12q14.2 61797598 61964327 166730 Amplification 2.65481 HB8 chr6 6p24.3-6p24.2 10443315 10616029 172715 Amplification 2.64576 HB8 chr1 1q42.2 229024410 229199429 175020 Amplification 2.82607 HB8 chr12 12q24.32 124916675 125091897 175223 Amplification 2.67604 HB8 chr1 1q44 241756423 241932247 175825 Amplification 2.57733 HB8 chr12 12p13.2 11851648 12028272 176625 Amplification 3.09241 HB8 chr6 6p21.1 43194752 43372930 178179 Amplification 2.50885 HB8 chr1 1q42.2 229447886 229626756 178871 Amplification 3.02352 HB8 chr12 12q13.13 50036882 50219473 182592 Amplification 3.02339 HB8 chr12 12p13.1 13725445 13908205 182761 Amplification 2.58072 HB8 chr17 17p13.2 4397439 4580283 182845 Amplification 2.53638 HB8 chr12 12p13.33 20703 205298 184596 Amplification 2.7938 HB8 chr12 12p13.2 12064206 12250007 185802 Amplification 2.82041 HB8 chr21 21q22.3 46478598 46666635 188038 Amplification 2.59921 HB8 chr17 17p12 11775479 11965405 189927 Amplification 2.67427 HB8 chr8 8q24.13 126408381 126599788 191408 Amplification 2.59701 HB8 chr12 12q21.2 76405655 76598675 193021 Amplification 2.54291 HB8 chr8 8q13.3 70933830 71128675 194846 Amplification 2.95626 HB8 chr1 1q21.3 151158441 151355224 196784 Amplification 2.59063 HB8 chr2 2q14.2 121304518 121501572 197055 Amplification 2.5403 HB8 chr2 2p13.3 70968248 71165486 197239 Amplification 2.5334 HB8 chr5 5q31.2 137537664 137735232 197569 Amplification 2.58002 HB8 chr12 12q13.13 51955473 52153836 198364 Amplification 3.10684 HB8 chr12 12q22 93087403 93286349 198947 Amplification 3.16058 HB8 chr12 12q24.31 124140243 124339413 199171 Amplification 2.71119 HB8 chr12 12q23.3 107031469 107231401 199933 Amplification 2.81013 HB8 chr5 5q33.1 150433141 150633197 200057 Amplification 2.52584 HB8 chr12 12q24.32 127064191 127264508 200318 Amplification 2.71693 HB8 chr12 12p12.1 24470330 24671686 201357 Amplification 2.64057 HB8 chr1 1q32.3 210896757 211098885 202129 Amplification 2.73674 HB8 chr12 12q24.22 115886346 116088678 202333 Amplification 2.74493 HB8 chr12 12q21.1 73154572 73357958 203387 Amplification 2.58493 HB8 chr12 12q21.1 70985826 71190602 204777 Amplification 2.6533 HB8 chr2 2p24.1 20355012 20559896 204885 Amplification 2.74519 HB8 chr12 12p11.21 31766504 31973334 206831 Amplification 3.08659 HB8 chr19 19p13.12 14153785 14362633 208849 Amplification 2.8454 HB8 chr12 12p13.1 13363470 13572745 209276 Amplification 2.87545 HB8 chr12 12q23.1 98767488 98976834 209347 Amplification 2.57617 HB8 chr20 20q13.2 49801099 50010559 209461 Amplification 2.54657 HB8
chr1 1p36.32 3767167 3978129 210963 Amplification 3.0744 HB8 chr1 1q44 242592361 242803893 211533 Amplification 3.27081 HB8 chr12 12p12.1 22419723 22631872 212150 Amplification 2.67382 HB8 chr12 12q23.1 98977416 99190709 213294 Amplification 3.05847 HB8 chr12 12q24.21 114602665 114816292 213628 Amplification 2.93213 HB8 chr16 16p13.11 15984507 16202763 218257 Amplification 2.59417 HB8 chr6 6p21.31 33823838 34042590 218753 Amplification 2.52393 HB8 chr12 12p11.21 30653622 30874050 220429 Amplification 2.74545 HB8 chr12 12q21.32 86405635 86626715 221081 Amplification 2.74776 HB8 chr2 2q12.3-2q13 108393695 108615041 221347 Amplification 2.7378 HB8 chr20 20q11.23 36240041 36462736 222696 Amplification 2.68005 HB8 chr12 12q23.1-12q23.2 99867320 100090573 223254 Amplification 2.96394 HB8 chr16 16p13.12 14397468 14621329 223862 Amplification 2.5177 HB8 chr12 12q21.1 71344014 71567938 223925 Amplification 2.74913 HB8 chr1 1q31.3-1q32.1 197370589 197594680 224092 Amplification 2.511 HB8 chr12 12q23.3 106799085 107023295 224211 Amplification 2.86147 HB8 chr17 17p12 13569112 13793528 224417 Amplification 2.50538 HB8 chr1 1q23.1 156141881 156367044 225164 Amplification 3.2123 HB8 chr6 6q21 107383082 107608538 225457 Amplification 2.60352 HB8 chr12 12q12 44127797 44353962 226166 Amplification 2.66136 HB8 chr1 1q42.2-1q42.3 232600622 232827424 226803 Amplification 2.78148 HB8 chr16 16q13-16q21 56666614 56894007 227394 Amplification 2.63887 HB8 chr12 12q24.23 117959314 118187059 227746 Amplification 2.61701 HB8 chr12 12q22 92537440 92766566 229127 Amplification 2.98902 HB8 chr1 1q25.1 173776359 174005890 229532 Amplification 3.0475 HB8 chr12 12q24.31 122406533 122637277 230745 Amplification 3.20633 HB8 chr21 21q22.3 41759088 41990151 231064 Amplification 2.55849 HB8 chr12 12q21.31 79083816 79314913 231098 Amplification 2.60188 HB8 chr12 12p13.31 6419011 6650595 231585 Amplification 2.79763 HB8 chr8 8q13.1 67338896 67570572 231677 Amplification 2.5099 HB8 chr17 17q24.1 60819003 61050861 231859 Amplification 2.61631 HB8 chr12 12q21.2 76602452 76836326 233875 Amplification 2.526 HB8 chr17 17q21.1-17q21.2 35574757 35809442 234686 Amplification 2.97996 HB8 chr1 1q44 242096151 242332867 236717 Amplification 2.7427 HB8 chr12 12q12 42194809 42431725 236917 Amplification 2.66747 HB8 chr1 1q31.1 184298372 184535342 236971 Amplification 2.73609 HB8 chr12 12p12.3 17480788 17718341 237554 Amplification 2.85782 HB8 chr2 2p24.1-2p23.3 23878419 24117450 239032 Amplification 2.63317 HB8 chr2 2p25.3 257017 496775 239759 Amplification 2.92375 HB8 chr12 12p12.1 22125592 22366107 240516 Amplification 3.05297 HB8 chr12 12q13.11 46561897 46804674 242778 Amplification 2.92845 HB8 chr1 1q32.1 200661593 200904972 243380 Amplification 2.68722 HB8 chr1 1q42.2 229201144 229445219 244076 Amplification 2.79887 HB8 chr2 2p25.3 2784 249862 247079 Amplification 2.52155 HB8
chr12 12q24.11 109895373 110146258 250886 Amplification 2.85165 HB8 chr1 1q32.3 210645039 210896174 251136 Amplification 2.92142 HB8 chr1 1q42.3 233858115 234109562 251448 Amplification 2.91515 HB8 chr12 12q24.11 109157939 109411000 253062 Amplification 2.99316 HB8 chr12 12p13.33 860318 1113411 253094 Amplification 2.91733 HB8 chr17 17p13.3 123705 377282 253578 Amplification 2.68928 HB8 chr1 1q25.1 172185466 172440257 254792 Amplification 2.97514 HB8 chr12 12p12.3 18003348 18260991 257644 Amplification 2.58647 HB8 chr1 1q32.1 202851846 203109971 258126 Amplification 2.82414 HB8 chr12 12q14.3 63934783 64193049 258267 Amplification 2.68548 HB8 chr12 12q23.2 101969404 102228014 258611 Amplification 2.56153 HB8 chr1 1q44 243522558 243781683 259126 Amplification 2.76604 HB8 chr2 2q36.3 228423522 228682888 259367 Amplification 2.5742 HB8 chr1 1q43 236594004 236853381 259378 Amplification 2.75878 HB8 chr1 1q21.3 152197979 152457532 259554 Amplification 2.53575 HB8 chr12 12q24.31 123876251 124136475 260225 Amplification 2.93021 HB8 chr2 2p25.1 11447413 11708143 260731 Amplification 3.12848 HB8 chr1 1q42.2 230648056 230910615 262560 Amplification 2.86335 HB8 chr1 1q44 245468659 245731998 263340 Amplification 2.93412 HB8 chr12 12q24.21 112999671 113263589 263919 Amplification 2.79183 HB8 chr19 19p13.11 16158145 16422580 264436 Amplification 2.63077 HB8 chr1 1q24.2 168596799 168861317 264519 Amplification 2.62427 HB8 chr1 1q25.3 179372354 179637067 264714 Amplification 2.93066 HB8 chr6 6p21.31 34042636 34307823 265188 Amplification 2.73339 HB8 chr16 16p11.2 29256954 29522235 265282 Amplification 3.07387 HB8 chr2 2p25.3 999448 1265744 266297 Amplification 2.76894 HB8 chr12 12q21.1 71572976 71839543 266568 Amplification 2.50605 HB8 chr2 2p24.3 13044794 13311441 266648 Amplification 2.82489 HB8 chr1 1q32.2 206281101 206548167 267067 Amplification 2.96573 HB8 chr12 12q14.2 62334672 62604479 269808 Amplification 2.63066 HB8 chr12 12q21.31 83486046 83756739 270694 Amplification 2.5247 HB8 chr12 12q15 67065845 67339521 273677 Amplification 2.62466 HB8 chr2 2q14.3 127662541 127937888 275348 Amplification 2.52827 HB8 chr12 12p12.1 24673940 24950048 276109 Amplification 2.76252 HB8 chr1 1q31.3 194346086 194622580 276495 Amplification 2.53164 HB8 chr17 17q25.3 75476961 75756044 279084 Amplification 2.52812 HB8 chr1 1q32.1 204186469 204467405 280937 Amplification 3.2427 HB8 chr12 12q14.2 62639721 62921430 281710 Amplification 2.86846 HB8 chr17 17q12 32753597 33035646 282050 Amplification 2.53063 HB8 chr12 12p12.3 17719380 18002893 283514 Amplification 2.50335 HB8 chr19 19q13.2 46442216 46728846 286631 Amplification 2.53393 HB8 chr1 1q32.3-1q41 211847444 212134081 286638 Amplification 2.64497 HB8 chr12 12q15 66777687 67065166 287480 Amplification 2.86226 HB8 chr2 2p23.3-2p23.2 27625536 27914602 289067 Amplification 2.53462 HB8
chr6 6p24.2 10619385 10908683 289299 Amplification 2.54661 HB8 chr1 1q42.13 226696490 226986623 290134 Amplification 2.87372 HB8 chr1 1q25.3 181811630 182102133 290504 Amplification 3.08588 HB8 chr16 16q23.3 81678221 81969208 290988 Amplification 2.52284 HB8 chr12 12p13.2-12p13.1 12551081 12842412 291332 Amplification 2.99694 HB8 chr12 12q23.1 96232454 96525456 293003 Amplification 2.59675 HB8 chr12 12q22 92766910 93060274 293365 Amplification 2.80489 HB8 chr12 12p11.21 31467208 31762106 294899 Amplification 2.86149 HB8 chr12 12q23.2-12q23.3 102348972 102643967 294996 Amplification 2.63527 HB8 chr12 12p13.31 8501367 8796557 295191 Amplification 2.76148 HB8 chr20 20p11.21 22399041 22694795 295755 Amplification 2.50657 HB8 chr12 12q14.1 56723841 57019782 295942 Amplification 2.95038 HB8 chr1 1q21.3 150857617 151155834 298218 Amplification 2.98491 HB8 chr12 12q24.13 111426294 111724589 298296 Amplification 3.17098 HB8 chr1 1q21.1-1q21.2 147788467 148087119 298653 Amplification 2.61276 HB8 chr12 12p13.2 12251090 12550932 299843 Amplification 3.00109 HB8 chr12 12q24.32 126371197 126671554 300358 Amplification 3.03392 HB8 chr1 1q31.2 191155911 191456542 300632 Amplification 2.97374 HB8 chr2 2q33.3 208378695 208679558 300864 Amplification 2.55105 HB8 chr1 1q41 217701931 218003177 301247 Amplification 2.88717 HB8 chr2 2p25.1 10003380 10305640 302261 Amplification 2.72071 HB8 chr2 2p22.1 38730556 39034690 304135 Amplification 2.61866 HB8 chr2 2q12.1-2q12.2 105174408 105479589 305182 Amplification 2.52622 HB8 chr1 1q41 212308532 212616001 307470 Amplification 2.86854 HB8 chr12 12p12.1 21309964 21618449 308486 Amplification 2.80162 HB8 chr12 12p13.2 11093660 11403818 310159 Amplification 3.12057 HB8 chr16 16q24.3 88281249 88592627 311379 Amplification 2.57919 HB8 chr2 2q12.2 106326031 106638833 312803 Amplification 2.5522 HB8 chr1 1q24.1 165126307 165439178 312872 Amplification 2.77878 HB8 chr10 10q26.3 133387412 133700329 312918 Amplification 2.51173 HB8 chr2 2q11.1 94751184 95064428 313245 Amplification 2.69526 HB8 chr12 12q21.33 87507419 87821402 313984 Amplification 2.5174 HB8 chr20 20q13.13 48622334 48936625 314292 Amplification 2.59324 HB8 chr12 12q13.13 51578241 51893022 314782 Amplification 3.35185 HB8 chr12 12q14.3 64195324 64511819 316496 Amplification 2.93145 HB8 chr5 5q33.1 149054778 149379208 324431 Amplification 2.50842 HB8 chr12 12q24.13-12q24.21 112515868 112840716 324849 Amplification 2.71028 HB8 chr20 20q12 38677568 39003941 326374 Amplification 2.50217 HB8 chr12 12q14.2 62000447 62332872 332426 Amplification 2.65171 HB8 chr1 1q42.12 223538053 223871157 333105 Amplification 2.99292 HB8 chr16 16p13.12-16p13.11 14623589 14957434 333846 Amplification 3.02067 HB8 chr1 1q32.2 206564873 206899931 335059 Amplification 2.87892 HB8 chr17 17q21.2-17q21.31 37772764 38108225 335462 Amplification 2.60189 HB8 chr1 1q25.3 179033332 179370273 336942 Amplification 2.86249 HB8