Identification of novel risk variants for sarcoma and other cancers by whole exome sequencing analysis in cancer cluster families DOI:

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1 Identification of novel risk variants for sarcoma and other cancers by whole exome sequencing analysis in cancer cluster families Jones, R. M. (2017). Identification of novel risk variants for sarcoma and other cancers by whole exome sequencing analysis in cancer cluster families DOI: /23/59f13ee5d5573 DOI: /23/59f13ee5d5573 Link to publication in the UWA Research Repository Rights statement This work is protected by Copyright. You may print or download ONE copy of this document for the purpose of your own non-commercial research or study. Any other use requires permission from the copyright owner. The Copyright Act requires you to attribute any copyright works you quote or paraphrase. General rights Copyright owners retain the copyright for their material stored in the UWA Research Repository. The University grants no end-user rights beyond those which are provided by the Australian Copyright Act Users may make use of the material in the Repository providing due attribution is given and the use is in accordance with the Copyright Act Take down policy If you believe this document infringes copyright, raise a complaint by contacting repository-lib@uwa.edu.au. The document will be immediately withdrawn from public access while the complaint is being investigated. Download date: 28. Apr. 2018

2 Identification of novel risk variants for sarcoma and other cancers by whole exome sequencing analysis in cancer cluster families Submitted by Rachel Jones This thesis is presented for the degree of Doctor of Philosophy The University of Western Australia School of Surgery 2017 i

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4 Declaration I, Rachel Jones, certify that: This thesis has been substantially accomplished during enrolment in the degree. This thesis does not contain material which has been accepted for the award of any other degree or diploma in my name, in any university or other tertiary institution. No part of this work will, in the future, be used in a submission in my name, for any other degree or diploma in any university or other tertiary institution without the prior approval of The University of Western Australia and where applicable, any partner institution responsible for the joint-award of this degree. This thesis does not contain any material previously published or written by another person, except where due reference has been made in the text. The work(s) are not in any way a violation or infringement of any copyright, trademark, patent, or other rights whatsoever of any person. The research involving human data reported in this thesis was assessed and approved by The University of Western Australia Human Research Ethics Committee. Approval number: RA/4/1/6434. Third party editorial assistance was provided in the preparation of the thesis by Dr Tegan McNab. iii

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6 For Gareth and Abbie... [A] knowledge of sequences could contribute much to our understanding of living matter. Frederick Sanger [1980] v

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8 Abstract Cancer is a genetic disease caused by an accumulation of genetic and epigenetic alterations. Cancers can be caused by mutations that arise in single somatic cells, resulting in sporadic tumours or mutations that occur in the germline, resulting in hereditary predisposition to cancer. While only a small proportion of cancers are estimated to involve an inherited genetic mutation, familial clustering of cancers is relatively common. More than 100 cancer predisposition genes have been identified using a variety of genetic strategies. However, only a small proportion of familial cancer risk can be explained by established cancer susceptibility genes. The identification of genes that predispose individuals to cancer is of high importance in human medical research as inherited genetic variants in genes that metabolise and process drugs can influence response to treatment. Sarcomas are a rare group of cancers that arise predominantly from the connective tissues of the body. Despite representing only 1% of all cancers, sarcomas are a high impact group of cancers that disproportionately affect the young. While it is sometimes difficult to distinguish sporadic from hereditary cancer, rare cancer, such as sarcoma, occurring twice within the one family is epidemiologically striking. The use of whole exome sequencing (WES) in families currently represents an optimal study design for the identification of rare genetic variants involved in the risk of cancer. Families in which multiple members develop a rare form of cancer, such as sarcoma, are more likely to have a mutation segregating in an inherited cancer gene compared to families affected by more common types of cancer. vii

9 In this study, three cancer cluster families (19 individuals) with a sarcoma proband were selected from the International Sarcoma Kindred Study, and WES was performed on germline DNA from both affected and unaffected family members using the Ion Proton platform at 100X coverage. WES data was annotated using Annotate Variation (ANNOVAR) and Regulome database (RegulomeDB). Putative structural and regulatory variants were filtered using genomic location and variant class or RegulomeDB score. Three different strategies were used to prioritise rare private variants, known rare variants and candidate gene variants. Association and segregation analyses of the prioritised variants were used to identify eight nominally significant germline risk variants in the ARHGAP39, C16orf96, ABCB5, ZFP69B, UVSSA, BEAN1, KIF2C and PDIA2 genes that show segregation with cancer in the families. Matched tumour and germline analyses were performed on WES data generated using the Illumina HiSeq 4000 at 60X coverage for two myxoid liposarcoma patients from two of the cancer cluster families. A total of 13 statistically significant somatic mutations were identified using VarScan2 and Strelka (PRMT5, ASPN, LAMA2, TET2, FHOD3, GATAD2A, ADSSL1, P4HTM, ABL1, SLC6A18, PLK2 and two intergenic variants between SLC22A20 and POLA2, and SDR16C6P and PENK). A region of loss of heterozygosity on chromosome 16 was also identified in one of the myxoid liposarcoma tumours. Whole genome sequencing (WGS) of germline DNA using the Illumina HiSeq X Ten platform was available for 561 sarcoma cases and 1,144 healthy ageing controls from the Garvan Institute for Medical Research. Using this WGS data, variant burden analyses were performed independently for summed nonsynonymous deleterious variants and putative regulatory variants to validate target regions identified in the cancer cluster families. The target regions were defined as the genes in which candidate germline and somatic mutations were identified and included 1,000 bases either side. For intergenic variants, both flanking genes were included. Of the 21 regions analysed, six (C16orf96, SLC6A218, TET2, ARHGAP39, ABL1 and a region encompassing SLC22A20 and POLA2 ) were found to have a significantly higher burden of variants in sarcoma cases compared to controls (p-value < 2.38 x 10 3 ). viii

10 The current study was the first to perform WES on cancer cluster families identified by a sarcoma proband. The results indicate the utility of this approach to identify novel sarcoma candidate risk genes by sequencing a small number of mixed cancer cluster families and validating the results in larger population cohorts. Genomic regions identified in this study should be prioritised for further studies to determine the role of these genes in cancer and sarcoma pathogenesis. ix

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12 Contents Declaration Abstract Table of contents List of tables List of figures Acknowledgements Authorship declaration Abbreviations iii vii xi xviii xx xxiii xxv xxvii 1 Literature review Cancer Cancer genetics Familial cancers Familial cancer predisposition syndromes Familial cancer clusters Evidence for pleiotropic genetic risk factors Sarcoma Sarcoma genetics Methods for identifying genetic risk variants Linkage mapping Association DNA sequencing Whole exome sequencing xi

13 1.6.5 Whole exome sequencing of cancer cluster families Next generation sequencing study considerations Known cancer predisposition genes Summary Aims Aim 1: Whole exome sequencing of three cancer cluster families identified by a sarcoma proband Introduction Ion Proton platform Methods Families selected for whole exome sequencing DNA extraction Whole exome sequencing Library preparation Exome sequencing Sequence alignment and variant calling Variation to sequence alignment and variant calling Torrent variant caller plugin Genome analysis toolkit Intersect variant calls from Torrent Variant Caller and Genome Analysis Toolkit Recalibrate variants Genotype concordance Results Families selected for whole exome sequencing Whole exome sequencing Variant calling Recalibrate variants Genotype concordance Discussion Evaluation of families used in this study The use of whole exome sequencing to identify disease causing variants Limitations of whole exome sequencing xii

14 2.4.4 The Ion Proton sequencing platform Base calling software Concordance Aim 2: Identification of candidate germline risk variants in three cancer cluster families Introduction Bioinformatic strategies for variant filtering and prioritisation in whole exome sequencing Annotation Annotation of non-coding regions Variant class filtering Population frequency filtering Evolutionary conservation Functional impact prediction Association analysis in families Familial segregation Outline of chapter Methods Ascertainment bias correction Intersection Annotation and filtration Prioritisation strategies Prioritisation using a rare private variants strategy Prioritisation using a known rare variants strategy Prioritisation using a candidate gene strategy Methods for testing association of variants with cancer phenotypes Bonferroni correction Familial segregation analysis Evidence further supporting candidate risk genes Results Variant prioritisation Prioritisation using a rare private variants strategy Prioritisation using a known rare variants strategy 59 xiii

15 Prioritisation using a candidate gene strategy Summary of annotated variants from each prioritisation strategy Rare private variants Association analysis in SOLAR Segregation analysis results Known rare variants Association analysis in SOLAR Segregation analysis results Candidate gene variants Association analysis in SOLAR Segregation analysis results Evidence further supporting germline risk genes Discussion Variant filtering and prioritisation strategies Association and segregation analyses of candidate risk variants in families The ABCB5 gene The KIF2C gene The PDIA2 gene Conclusion Aim 3: A comparison of matched tumour and germline DNA from two sarcoma patients Introduction Myxoid liposarcoma Somatic variants Loss of heterozygosity Somatic copy number alteration Bioinformatic assessment of matched tumour and germline samples Somatic mutations and drug sensitivity Outline of chapter Methods Whole exome sequencing xiv

16 4.2.2 Pre-processing and quality control Adapter trimming Sequence alignment and calling BAM quality control Generate mpileup file Somatic variant calling using VarScan Somatic variant calling using Strelka Evidence further supporting somatic risk genes Drug sensitivity Loss of heterozygosity variant calling using VarScan Variant annotation and filtering Somatic copy number analysis using VarScan Results Whole exome sequencing Sequence alignment and calling BAM quality control Somatic variant calling VarScan Validation of somatic variants using Strelka Evidence further supporting somatic risk genes Drug sensitivity Loss of heterozygosity variants Copy number analysis Discussion Comparison of results in the context of published literature on myxoid liposarcoma genetics Strengths Limitations Summary Aim 4: Variant burden analyses at candidate risk loci in sarcoma cases and healthy ageing controls Introduction Variant burden analyses in sarcoma cohorts Methods xv

17 5.2.1 Study participants Whole genome sequencing Genomic regions selected for validation Statistical analyses Results Identification of nonsynonymous deleterious variants in the target regions Statistical analyses Nonsynonymous deleterious variants Putative regulatory variants Discussion Novel findings Known cancer genes Clinical implications Strengths and limitations Conclusion Conclusion Summary of results Clinical utility of findings Review of methodology Recommendations for future work Bibliography 149 Appendices 239 A World Health Organisation classification of soft tissue tumours and bone tumours 241 B Novel tumour-predisposing genes identified by whole exome sequencing 251 C Familial cancer syndromes associated with sarcomas 265 D Translocations associated with sarcomas 271 xvi

18 E Genetically complex sarcomas 277 F Known cancer predisposition genes 281 G Candidate genes used for variant prioritisation based on a priori knowledge of cancer biology 289 H Genes in which variants were also prioritised using the candidate gene prioritisation strategy 293 I Patient 1-II-2: Copy number variation by chromosome 297 J Patient 2-II-1: Copy number variation by chromosome 303 K A list of nonsynonymous deleterious variants included in variant burden analyses 309 L Gene identified by variant burden analyses by Ballinger et al. (2016) and Brohl et al. (2017) 315 M A list of putative regulatory variants included in variant burden analyses 319 xvii

19 List of Tables 2.1 Parameters used to create whole exome sequencing run plans using Torrent Suite software Parameters used to run the Torrent Variant Caller plugin to call bases Parameters used for Genome Analysis Toolkit UnifiedGenotyper to call bases Depth of coverage summary from Torrent Suite Genome Analysis Toolkit VariantRecalibrator tranche results Discordant genotype calls between the Agilent HaloPlex custom panel and whole exome sequencing for Patient 2-II Discordant genotype calls between the Agilent HaloPlex custom panel and whole exome sequencing for Patient 3-III Classification of Regulome database scores Functional annotation of intersect file using ANNOVAR Summary of variant annotation using Annotate Variation and Regulome Database for each prioritisation strategy Summary of SOLAR association results for rare private variants Summary of SOLAR association results for known rare variants Summary of SOLAR association results for candidate gene variants Summary of findings from in silico resources investigating the role of candidate germline risk variants in cancer pathogenesis Summary of search results from PubMed for genes in which germline variants were identified Parameters specified for VarScan2 somaticfilter to filter false positives from the high confidence somatic mutations xviii

20 4.2 Raw data summary from Macrogen Inc. for Patient 1-II-2 and Patient 2-II-1 germline and tumour samples Summary statistics generated using Samtools flagstat for Patient 1-II-2 and 2-II-1 germline and tumour samples Results from VarScan2 somaticfilter to remove possible false positives from the high confidence somatic calls for Patient 1-II-2 and Patient 2-II Somatic variants identified by VarScan2 and Strelka for Patient 1-II Somatic variants identified by VarScan2 and Strelka for Patient 2-II Summary of findings from in silico resources investigating the role of somatic risk variants and the genes in which they arise in cancer pathogenesis Summary of search results from PubMed for genes in which somatic variants were identified Statistically significant high confidence loss of heterozygosity variants for Patient 1-II Genomic coordinates for target regions in which germline and somatic risk variants were identified Classification of Regulome database scores Annotated summary of nonsynonymous deleterious variants and putative regulatory variants in the target regions Odds ratios, p-values and 95% confidence intervals from Fisher s exact test for target regions for nonsynonymous deleterious variants Odds ratios and p-values from Fisher s exact test for target regions for putative regulatory variants xix

21 List of Figures 1.1 Location of known cancer predisposition genes Pedigree of family Pedigree of family Pedigree of family Whole exome sequencing pipeline flowchart The number of variants called by Torrent Variant Caller and Genome Analysis Toolkit UnifiedGenotyper, and the number of variants that were called by both callers (intersect) Genome Analysis Toolkit VariantRecalibrator tranche plot Genome Analysis Toolkit VariantRecalibrator projection for mapping quality rank sum (MQRankSum) versus haplotype score Concordance of genotype calls between the Agilent HaloPlex custom panel and whole exome sequencing on Ion Proton for three patients Genotypes for the ARHGAP39 variant that shows segregation in patients with cancer in family Genotypes for the C16orf96 and ABCB5 variants that show segregation in patients with cancer in family Genotypes for the ZFP69B, BEAN1, UVSSA and KIF2C variants that show segregation in patients with cancer in family Genotypes for the PDIA2 variant that shows segregation in patients with cancer in family Pedigree of family 1 highlighting sarcoma Patient 1-II-2 for tumour-germline comparison Pedigree of family 2 highlighting sarcoma Patient 2-II-1 for tumour-germline comparison xx

22 4.3 Genome analysis toolkit depth of coverage summary for Patient 1-II-2 and Patient 2-II-1 germline and tumour DNA Insert size histogram plots generated by Picard for Patient 1-II-2 and Patient 2-II-1 germline and tumour samples Pedigree of family 1 indicating genotypes for each patient at chr16: (rs ) in the RBL2 gene xxi

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24 Acknowledgements I would like to acknowledge support from Mandy Basson and the Board of Directors of the Abbie Basson Sarcoma Foundation Ltd (Sock it to Sarcoma!). I would like to sincerely thank David Thomas, Mandy Ballinger and Mark Pinese, for providing the DNA samples and data used in this thesis. I would also like to acknowledge the participants from the International Sarcoma Kindred Study and the Medical Genome Reference Bank. I would like to express my gratitude to my supervisors Eric Moses, Phillip Melton, David Wood, David Thomas and Evan Ingley, for their guidance and for the opportunity to pursue this project. I would also like to acknowledge Jane Allen and Barry Iacopetta for their support. I would like to thank all my friends at the Centre for Genetic Origins of Health and Disease for your daily guidance and support, especially Alex Rea for his assistance in the lab and Gemma Cadby for her helpful advice and for reading drafts. I would also like to thank Tegan McNab for proofreading my thesis. I am grateful to my family and friends who have always supported my studies. xxiii

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28 Abbreviations Abbreviation *.bam *.bed *.sam *.vcf ABC Alt ANNOVAR ASPREE ATP ATPase ATRA B BCFtools BWA BWA-MEM Chr CNV COSMIC CpG CREB Definition Binary Alignment/Map Browser Extensible Data Sequence Alignment/Map Variant Call Format ATP-binding cassette Alternate allele Annotate Variation ASPirin in Reducing Events in the Elderly Adenosine Triphosphate Adenosinetriphosphatase All-Trans-Retinoic-Acid Benign Binary Variant Call Format Tools Burrows-Wheeler Aligner Burrows-Wheeler Aligner Maximal Exact Matches Chromosome Copy Number Variation The Catalogue of Somatic Mutations in Cancer 5 C phosphate G 3 camp Response Element-binding Protein xxvii

29 Abbreviation Definition D Deleterious dbsnp Short Genetic Variations Database DNA Deoxyribonucleic Acid DNase Deoxyribonuclease dntp Deoxynucleotide E2F E2 Factor ECM Extracellular Matrix ENCODE Encyclopedia of DNA Elements eqtl Expression Quantitative Trait Loci ER Endoplasmic Reticulum ERbB Erythroblastosis ERK Extracellular Signal-Regulated Kinase ESC Embryonic Stem Cells ExAC Exome Aggregation Consortium FAMMM Familial Atypical Multiple Mole Melanoma FFPE Formalin-Fixed and Paraffin-Embedded GATK Genome Analysis ToolKit GeneRIF Gene References into Functions GERP Genomic Evolutionary Rate Profiling GO Gene Ontology GOHaD Centre for Genetic Origins of Health and Disease GPCR G Protein Coupled Receptor GTP Guanosine Triphosphate GTPase Guanosine Triphosphatase GWA Genome Wide Association HapMap International Haplotype Project HDI Histone Deacetylation Inhibitor hg19 Human Genome build 19 xxviii

30 Abbreviation hmscs IG IGV INDEL Int isec ISKS Kb LOD LOH MAF MGRB MPNST MQRankSum mrna NCBI NGS NS NTR OMIM P PDI PNET PolyPhen-2 Probit Q QC Rb Definition Human bone marrow-derived Mesenchymal Stromal Cells Intergenic Integrative Genomics Viewer Insertions and Deletions Intronic BCFtools Intersect International Sarcoma Kindred Study Kilobase Logarithm of the Odds Loss Of Heterozygosity Minor Allele Frequency Medical Genome Reference Bank Malignant Peripheral Nerve Sheath Tumour Mapping Quality Rank Sum Messenger Ribonucleic Acid National Center for Biotechnology Information Next Generation Sequencing Nonsynonymous Neurotrophins Online Mendelian Inheritance in Man Possibly damaging Protein Disulphide Isomerase Primitive Neuroectodermal Tumour Polymorphism Phenotyping-2 Probability Unit Base Quality Score Quality Control Retinoblastoma xxix

31 Abbreviation Ref RegulomeDB RNA Robo rs ID S SCNA SIFT SLBP SNP SNV SOLAR T TF TMAP TVC UCSC USA UTR UTR3 UTR5 UWA VQSLOD VQSR WES WGS Definition Reference allele Regulome Database Ribonucleic Acid Roundabout family of proteins Reference SNP Identification Synonymous Somatic Copy Number Alteration Sorting Intolerant from Tolerant Stem-Loop Binding Domain Single Nucleotide Polymorphism Single Nucleotide Variant Sequential Oligogenic Linkage Analysis Routines Tolerated Transcription Factor Torrent Mapping Alignment Program Torrent Variant Caller University of California Santa Cruz United States of America Untranslated Region 3 Untranslated Region 5 Untranslated Region The University of Western Australia Variant Quality Score Log-Odds Variant Quality Score Recalibration Whole Exome Sequencing Whole Genome Sequencing xxx

32 Chapter 1 Literature review 1.1 Cancer Collectively, cancers are a diverse spectrum of human diseases with a common progression resulting from the failure to regulate normal cell growth, proliferation and apoptosis. 1 Cancers can arise from any of the cell or tissue types in the human body and are classified accordingly. 2 The most common cancers in adults are carcinomas, (approximately 90% of cancers) 2 which are derived from epithelial cells that line body cavities and glands. 3 Lymphomas and leukaemias arise in the tissue that gives rise to lymphoid and blood cells and account for approximately 8% of human malignancies. 3, 4 Melanomas, retinoblastomas, neuroblastomas and glioblastomas are derived from dividing cells in melanocytes, ocular retina, neurons and neural glia, respectively. 3 Sarcomas arise from the connective tissues such as bones, tendons, cartilage and fat. 2 Cancer is one of the leading worldwide causes of death with over 14 million people affected each year. 5 In 2012, there were 4.3 million premature deaths from cancer with premature deaths expected to increase 44% from 2012 to , 7 The lost years of life and productivity caused by cancer represent the largest cost to the global economy compared to other causes of death. 8 1

33 1.2 Cancer genetics Cancer is a genetic disease arising from an accumulation of genetic and epigenetic mutations. 9 These mutations can deregulate multiple complex regulatory pathways of genes affecting cellular growth, division, migration, and survival. 10 Tumour genomes usually exhibit many mutations and can be highly unstable. 11 Mutations can range from intragenic mutations to large gains and losses of chromosomal material. 9 A genetic mutation is a permanent change in the DNA sequence. A polymorphism is a genetic variation that is common in the population. The arbitrary cut-off between a mutation and a polymorphism is 1%, that is, the less common allele of a polymorphism must have a frequency of at least 1% in the population. 12 Mutations in a cancer genome can comprise the following types of DNA change: substitutions, insertions or deletions of small or large segments of DNA, rearrangements, copy number increases, and copy number reductions. 13 Cancer cells can also acquire new DNA sequences from viruses including human papillomavirus, Epstein-Barr virus, hepatitis B virus, human T-lymphotropic virus 1, and human herpesvirus. 14 Cancer genomes can also acquire epigenetic changes which alter chromatin structure and gene expression. 15 There can be anywhere between tens to thousands of mutations per cancer genome. 16 The substantial variation in the number and pattern of mutations in individual cancers reflects exposure to different risk factors, DNA repair defects, and the cellular origins. 17 Mutations that occur in cancers fall into two functional categories: mutations required for tumourigenesis, and mutations that merely occur during tumourigenesis and do not contribute to the process. These are called driver and passenger mutations, respectively. Drivers confer a selective advantage during clonal evolution and therefore drive the tumourigenesis process. Passenger mutations do not appear in tumours as a result of evolutionary selection, but rather as a variation that occurs by chance in a cell that harbours a driver mutation. It is likely that most cancers carry 2

34 more than one driver mutation, and the number of drivers varies between cancer 13, 16, type. Mutations can arise in three broad categories of genes - oncogenes, tumour suppressor genes, and genome stability genes. Mutations in oncogenes and tumour suppressor genes drive the tumourigenesis process by increasing proliferation or inhibiting apoptosis, respectively, whereas mutations in genome stability genes drive tumourigenesis by increasing the rate of mutations in other genes. 9 The characterisation of these genes has led to the discovery of the biochemical pathways underlying the process of tumourigenesis, and also to a better understanding of the normal homeostatic roles these pathways play in healthy cells and tissues. 21 Mutations in these three classes of genes can occur in single somatic cells, resulting in sporadic tumours, or in the germline, resulting in hereditary predisposition to cancer. Sporadic cancers develop due to mutations that arise during a person s lifetime. The majority of cancers (90-95%) develop sporadically due to genetic mutations that result from DNA damage from exposure to environmental and lifestyle factors. 22 Environmental risk factors include occupational exposures (chemicals, dust, and industrial processes), sunlight, radiation, and environmental pollution. 23 Lifestyle factors that may increase the risk of developing cancer include smoking, excessive alcohol consumption, poor diet, obesity and physical inactivity, chronic 24, 25 infections, sun tanning, and sunburn. Only a small proportion (5-10%) of cancers are estimated to involve an inherited genetic mutation However, familial clustering of cancers is relatively common. 31 Familial clustering is the occurrence of a disease, such as cancer, in some families more than what would be expected from the presence in the general population. 32 Familial clustering of cancer can be measured by familial proportion (the proportion of cases with an affected relative), which has been reported as high as 20% in prostate cancer. 33 Familial clustering of cancers is likely due to a combination of environmental factors, rare gene mutations with high penetrance and more common, lower penetrant gene variants that act together to increase cancer 32, susceptibility. 3

35 1.3 Familial cancers All cancers, both rare and common, show some degree of familial clustering. 37 Cancers can be two- to four-fold more common in first degree relatives of individuals with cancer Familial cancer predisposition syndromes Familial clustering of cancers can sometimes represent a familial cancer predisposition syndrome. A familial cancer predisposition syndrome manifests when multiple members of a family inherit gene mutations that predispose them to one or more types of cancer. 39 These families have multiple affected individuals, and family members often show early onset of cancer, multiple primary sites of disease, and occasionally bilateral involvement of paired organs. 35, 39 Some cancer predisposition syndromes appear to confer an increased risk of adult-onset cancers, such as breast, ovarian and colorectal cancers Other syndromes increase the susceptibility of tumour onset in childhood, such as hereditary retinoblastoma, 43 or early onset in both children and adults, such as von Hippel-Lindau disease. 44 Most familial cancer predisposition syndromes are transmitted in a Mendelian autosomal dominant manner. 35, 45 Dominant mutations require only one defective allele to be present for the individual to be predisposed to cancer. Individuals with one defective and one normal allele are heterozygous. An example of a Mendelian autosomal dominant cancer predisposition syndrome is hereditary breast-ovarian cancer. 46 This syndrome is caused by mutations in the BRCA1 and BRCA2 genes. 47 Women with germline mutations in BRCA1 have a 46 65% risk of developing breast cancer by age 70, while those with a BRCA2 mutation 40, 48 have a lower risk of 43 45% by age 70. Less often, familial cancer predisposition syndromes can be transmitted in an autosomal recessive manner. In the case of recessive mutations, both alleles must be mutated for the individual to have a predisposition to cancer. Individuals who inherit a recessive germline mutation in a gene are known as carriers and carry the mutation in every cell of their body. There is a variable risk that a carrier will develop cancer. A carrier will not develop cancer unless the remaining 4

36 normal allele is also mutated. The particular mutation, other genes, and dietary, lifestyle and environmental factors can influence risk. 49 The likelihood that a carrier will develop cancer is defined as the penetrance of the mutation. 3 An example of an autosomal recessive cancer predisposition syndrome is xeroderma pigmentosum complementation group A, characterised by increased sensitivity to sunlight with the development of carcinomas at an early age. 50 Xeroderma pigmentosum complementation group A has been associated with homozygous or compound heterozygous mutations in the XPA gene. 50 The study of familial cancer predisposition syndromes has led to the identification of genes critical to carcinogenesis and has also informed our understanding of the fundamental biology of human cancer. 51 Li and Fraumeni (1969) described the first familial cancer syndrome in four unrelated children with sarcoma and other affected family members. 52 They hypothesised that the occurrence of various malignancies in a family might represent a familial cancer syndrome due to the transmission of an autosomal dominant gene mutation. 52 In 1990 the TP53 gene was identified as the underlying gene responsible for Li-Fraumeni syndrome. 53 The TP53 gene encodes a tumour suppressor protein that responds to diverse cellular stresses to regulate expression of target genes, thereby inducing cell cycle arrest, apoptosis, senescence, DNA repair, or changes in metabolism Germline mutations in the TP53 gene were later established to also be the underlying genetic cause for many other malignancies Familial cancer clusters There are also familial cancer clusters that are not defined by known hereditary cancer syndromes. Familial cancer clusters are those that do not exhibit the features of hereditary types of cancer but occur in more individuals in the family than statistically expected. 36 In addition to familial clustering for the majority of specific cancers, aggregation of different types of cancers in families has also been observed. For example, individuals with BRCA1 and BRCA2 mutations, have not only increased susceptibility to breast and ovarian cancers, but also colon, cervix, uterus, pancreas, and prostate cancers. 57 5

37 1.4 Evidence for pleiotropic genetic risk factors Early studies assessed the discordant clustering of cancer in families to determine if there was a general susceptibility to cancer. Case-control, registry- and population-based studies have evaluated familial clustering using risk ratio and kinship coefficient estimations. 58 Identifying shared genetic associations between diseases (pleiotropy) is a useful approach to identify new risk loci, and may elucidate common aetiologies and help in risk prediction. 59 The largest studies using the Utah Population and Cancer Registry Database and the Swedish Family-Cancer Database demonstrated excess familial clustering at almost every cancer site in the body. 34, However, these studies focused on familial clustering exclusively in nuclear families, therefore, they were not able to separate the role of shared environmental and genetic factors in the familial aggregation of cancer. A more extensive study by Cannon-Albright et al. (1994) used the Utah Population Database to evaluate familial clustering for more distant relatives. 60 As familial risk can be due to shared exposure to an environmental risk and/or a common genetic mutation, examination of familial clustering in near and distant relatives is useful. In more distant relationships, shared familial environment might be less likely, and the probability of shared genotypes can be measured. 60 This study found that there was significant clustering of cancer outside the nuclear family for cancer sites. 60 These results support the hypothesis of an inherited basis to cancer of almost all sites and support the existence of more than one susceptibility locus for some cancers. 60 In support of this finding, a study by Amundadottir et al. (2005) analysed familial aggregation of cancer in extended families from Iceland to search for genetic factors that contribute to cancer at one or more sites in the body. 58 The authors found that most cancer sites demonstrated a significantly increased risk for the same cancer beyond the nuclear family. 58 They also found significantly increased familial clustering between different cancer sites in both close and distant relatives. 58 Therefore, Amundadottir et al. concluded that genetic factors are involved in the aetiology of many cancers and that these factors are in some cases shared by different cancer sites. 58 These findings support the conclusions by Cannon-Albright et al. However, shared environment or non-random mating for certain risk factors 6

38 also play a role in the familial clustering of cancer. 58 Several types of study designs can be used to identify genetic risk variants that may be involved in the aetiology of cancers. 1.5 Sarcoma Sarcomas are a rare group of cancers that arise predominantly from the embryonic mesoderm (the connective tissues of the body), for example, bones, muscles, cartilage and fat. There are over 70 different subtypes of sarcoma that are grouped into two broad classifications of bone or soft tissue (Appendix A). 64 The majority of sarcomas arise in the soft tissue, while malignant bone tumours make up just over 10% of all sarcomas. 65 Soft tissue sarcomas are often further sub-categorised by the line of differentiation, for example, liposarcoma (fat), leiomyosarcoma (smooth muscle), rhabdomyosarcoma (skeletal muscle) and fibrosarcoma (connective tissue). 66, 67 Bone tumours are further classified into bone-forming tumours, cartilage-forming tumours, marrow tumours, or vascular tumours. 68 It can be difficult to diagnose and classify this diverse group of malignancies with overlapping histological features. However, it is important to correctly determine the specific 67, 68 histologic subtype for management and treatment decisions. Sarcomas are a high impact group of cancers that disproportionately affect the young. Although sarcomas are rare, they contribute significantly to the burden of disease as they tend to affect teenagers and young adults. 69, 70 Sarcomas represent only 1% of all cancers in adults but represent 10% of cancers in children and 8% of cancers in adolescents and young adults. 71 There are approximately 800 new sarcoma cases in Australia each year Sarcoma genetics There is evidence to suggest a strong genetic basis to sarcomas. First, sarcomas disproportionately affect the young, with early age at diagnosis associated with 73, 74 a genetic basis for many heritable diseases, including hereditary cancers. Second, sarcomas are over-represented among survivors of melanoma, breast cancer, thyroid cancer, Hodgkin s lymphoma, and leukaemias. 75 Third, sarcoma survivors are at increased risk of secondary cancers. 76 Finally, several rare genetic 7

39 syndromes are associated with sarcomas such as Li-Fraumeni syndrome. 35 Appendix C contains a summary of hereditary syndromes associated with sarcoma including genes and genomic locations. In addition to sarcomas being associated with familial cancer predisposition syndromes, 52, sarcomas also show evidence of familial clustering. Up to 33% of paediatric sarcomas are estimated to be associated with a significant family history of cancers. 80 The risk of sarcomas is increased six-fold in relatives of children with sarcoma compared to age-matched controls. When a causal gene mutation is identified, this risk increased to over 250-fold. 81 Whilst some sarcomas are associated with familial inherited predisposition, most sarcomas do not have a known cause. Very little is currently known about the causes of sarcoma because they are so rare. 65 Several risk factors have been associated with sarcomas including ionising radiation, 82, 83 viruses (Epstein-Barr virus 84 and Kaposi s sarcoma-associated herpes virus 85 ), occupation, exposure to chemicals, hormones, 97, 98 antibiotics, 99 medications for nausea used during pregnancy, 100 use of antibiotics in babies, 101 birth weight, 102 gestational age, , 105 birth order, and maternal age. Sarcomas that arise due to somatic mutations are classified into two main groups based on genetics: 1. Sarcomas with specific recurrent genetic mutations on a background of relatively few other chromosomal changes 2. Sarcomas with no specific genetic mutations on a complex background of numerous chromosomal changes Approximately one-third of all sarcomas have specific recurrent genetic mutations. 106 These tumours either contain disease-specific chromosome translocations or specific activating mutations. Most sarcomas with specific recurrent genetic mutations are characterised by balanced or reciprocal translocations (the exchange of pieces between two chromosomes), resulting in two derivative chromosomes with no net gain or loss of chromosomal 8

40 material. 107 In some cases, only one derivative chromosome is formed, and some genetic material is lost. The fusion proteins produced as a result of the translocation can contribute to oncogenesis by increasing cell proliferation, promoting anchorage-independent cell growth, overriding cell contact adhesion, inhibiting apoptosis, enhancing invasion and suppressing terminal differentiation. 107 Appendix D contains a table of known translocations that have been associated with sarcoma. The remaining sarcomas in the specific recurrent genetic mutations group are characterised by specific activating mutations. 108 These tumours show some degree of aneuploidy, but generally, have less disordered karyotypes than the complex group of sarcomas. 83 An example of a sarcoma subtype with a specific activating mutation is gastrointestinal stromal tumours which have activating 109, 110 mutations in KIT or PDGFRA. The remaining two-thirds of sarcomas have highly complex unbalanced karyotypes lacking specific genetic translocations. 66, 111 This group is mostly composed of spindle cell or pleomorphic sarcomas including leiomyosarcoma, myxofibrosarcoma, pleomorphic liposarcoma, pleomorphic rhabdomyosarcoma, malignant peripheral nerve sheath tumour, angiosarcoma, extraskeletal osteosarcoma and spindle cell/pleomorphic unclassified sarcoma (previously known as spindle cell/pleomorphic malignant fibrous histiocytoma). 111 These neoplasms show gains and losses of many chromosomes or chromosome regions and amplifications. 111 Many of them share recurrent aberrations (such as the gain of 5p13-p15) that play a significant role in tumour progression or metastatic dissemination. 111 Appendix E lists the genomic regions identified in complex sarcomas. 1.6 Methods for identifying genetic risk variants Several study designs can be employed to identify genetic risk variants. Each study design is suited to identifying different types of mutations from highly penetrant genes in rare Mendelian disorders to low-penetrant variants in more common disease, and rare variants. 9

41 1.6.1 Linkage mapping Linkage mapping in families has been used with success in localising highly penetrant disease-causing genes (e.g., BRACA1 and BRACA2 ) and, in particular, those involved in rare Mendelian human diseases (e.g., Online Inheritance In Man (OMIM), Linkage analysis in families is a form of positional cloning and makes no underlying assumptions about the nature of the genes involved. In human disease studies, the aim of linkage mapping is first to determine the chromosomal location of putative risk genes by identifying polymorphic DNA markers that cosegregate with a disease of interest. The genes in such linkage regions are referred to as positional candidate risk genes. These genes are then prioritised for further genetic and molecular analyses to identify the specific causal mutations or polymorphisms. Linkage mapping has been used to identify highly penetrant susceptibility alleles associated with Mendelian familial cancer predisposition syndromes However, these variants explain only a small fraction of the genetics of all cancer cases. For example, inherited mutations in the BRCA1 and BRCA2 genes account for approximately 2% 3% of all breast cancer cases. 118, 119 However, more prevalent founder mutations in these genes can explain up to about 10% of the disease in 47, some populations Association It has been postulated that more common cancers that do not show a clear pattern of inheritance are caused by many genes that confer a small risk of disease. 124 For disease risk genes of small effect, association studies can be more powerful than linkage studies. 125 With the advent of dense panels of single nucleotide polymorphism (SNP) markers and high-throughput technology for efficiently genotyping them in thousands of individuals, the genome wide association (GWA) analysis study design was subsequently adopted widely for the genetic analysis of common human diseases. GWA is also a form of positional cloning and relies on linkage disequilibrium, the non-random association of alleles at different loci that is a function of population history. 10

42 GWA studies have identified thousands of lower penetrance risk variants for common human traits and diseases typically with small effect size (odds ratio between 1.1 and 1.5). 126 Lower penetrance genetic variants associated with non-familial syndrome breast cancer confer slight risk alterations (odds ratio of approximately 1.2), 127 compared to the high penetrance variants in BRCA1 and BRCA2 identified by linkage with odds ratios between 2 and Most genetic cancer risk variants identified so far confer relatively small increments in risk and explain only a small proportion of familial clustering. 128 The inability of the risk variants detected by GWA studies to account for much of the heritability of most common disorders, missing heritability, has led to an emerging view that rare variants with larger effect sizes could be responsible for a substantial proportion of genetic risk for complex human disease. 129 Significant advances in genome sequencing have now offered the possibility of using this technology as an alternative study design to GWA studies for the detection of rare genetic risk variants DNA sequencing DNA sequencing analysis is the process of determining the precise order of nucleotides in a given DNA sample. One aim of DNA sequencing is to identify genomic variations and to associate those changes with human disease. A breakthrough in DNA sequencing technology was the development of Sanger s chain termination method. 130 In this approach, sequencing occurs by the selective incorporation of a single chain-terminating dideoxynucleotide by DNA polymerase. 130 For approximately 40 years Sanger sequencing was the most widely used approach. Since the completion of the Human Genome Project in 2003, there have been substantive developments in Next Generation Sequencing (NGS) technologies. Whereas the first human genome took 13 years and several billion dollars to complete, a human genome can now be sequenced in a day for $1,000 US (at 20X coverage). The speed of sequencing has increased as NGS enables the simultaneous detection of multiple mutations in multiple genes by the parallel sequencing of millions of different DNA fragments. 131 The development of affordable and efficient next generation DNA sequencing technologies has now provided a new 11

43 study paradigm to search for rare risk variants involved in common, complex diseases. The impact of NGS technology on the discovery of genetic variants in human disease has been profound. Since the introduction of NGS there have been enormous advances in speed, read length and throughput of sequencing studies. 132 The advent of NGS has allowed the inquiry of nearly every base in the genome. 133 The growth in cancer genomics discovery has been unprecedented; knowledge of genes frequently mutated in cancer has grown from four genes in 2004 to over 600 genes listed in the Catalogue of Somatic Mutations in Cancer (COSMIC) currently (v79, released 14-NOV-16). 134 Initiatives such as the Cancer Genome Atlas 135 and the International Cancer Genome Consortium, 136 have employed NGS strategies to characterise tumour genomes and provide multi-platform data for thousands of tumours from a variety of cancer types and subtypes. 137 Typical NGS applications include DNA sequencing, RNA sequencing (to measure gene expression changes to discover new transcripts), chromatin immunoprecipitation sequencing (to detect genome wide transcription factor binding sites and chromatin-associated modifications) and methylation sequencing (to profile various types of DNA methylation). 138, 139 Next generation DNA sequencing can be used for whole genome sequencing (WGS), whole exome sequencing (WES), or sequencing of a specifically targeted region of the genome. 138 The NGS workflow consists of multiple steps including library preparation and enrichment, sequencing, base calling, sequence alignment and variant calling Whole exome sequencing WES involves sequencing only the protein-coding region of the genome. The human exome makes up approximately 1% of the human genome. However, the majority (85%) of disease-causing mutations in Mendelian disorders are expected to arise in the exome. 140 Therefore WES is a cost-effective initial strategy to identify disease-causing variants. In the last decade, WES of unrelated individuals or families with multiple affected members a rare disorder has identified the genetic basis of diseases such as Freeman-Sheldon syndrome, Kabuki syndrome, Miller syndrome, and autosomal dominant spinocerebellar ataxia WES 12

44 studies have also identified more than 50 novel tumour-predisposing genes, listed in Appendix B Whole exome sequencing of cancer cluster families While WES has been used with great success to identify novel tumour predisposing mutations, only one published study has used WES to identify pleiotropic genetic risk variants that predispose families to more than one type of cancer. The recent WES study by Thutkawkorapin et al. (2016) utilised NGS technology to investigate a family with a dominant cancer syndrome with a high risk of both rectal and gastric cancer. 147 The authors hypothesised that the mixed representation of rectal and gastric cancer among family members was due to one predisposing mutation in one gene. 147 The authors performed WES in three family members, two with rectal cancer and one with gastric cancer, and followed up with WES and Sanger sequencing in additional family members, other patients and controls. 147 Thutkawkorapin et al. identified 12 novel nonsynonymous single nucleotide variants (SNVs) shared among five affected members of this family. The authors suggested that at least five of the 12 variants may be candidates that contributed to the disease in the family. 147 These variants did not segregate in other families and are therefore unlikely to be highly penetrant variants. 1.7 Next generation sequencing study considerations NGS technologies can be used to identify rare variants in tumour or germline DNA that increase an individual s susceptibility to developing cancer. 133 It is essential to compare tumour DNA with matched germline DNA to determine somatic and germline alterations in cancer. 148 Germline variants exist in the normal germline sequence. 149 Somatic variants are those in the tumour sequence but not in the normal germline sequence. 149 The ability of NGS to detect somatic variants depends on the variant frequency within the tumour sample, sample contamination, tumour heterogeneity, sequencing error, and the scarcity of somatic 150, 151 mutations within a genome. Recently there has been a return to family-based designs to identify rare risk variants involved in common human disease, based on the hypothesis that affected 13

45 133, members of the same family will carry the same rare susceptibility variant. Therefore, the number of individuals needed for rare variant discovery is potentially smaller than in cohorts of unrelated individuals. 133 Families used in these types of studies to identify rare inherited variants can either be consanguineous families, or non-consanguineous, large multigenerational and multiplex pedigrees. 133 Targeted sequencing technologies have been used to successfully identify new causal genes in hereditary non-polyposis colon cancer and familial adenomatous polyposis, 156 and hereditary breast and ovarian cancers. 157 Two-phase NGS family study designs are recommended. In the first phase, family members are sequenced, and the discovered variants are ranked according to their likelihood of being associated with the trait. 158 In the second phase, the variants are tested for association in an independent population-based sample. 158 Families used to study cancer clustering should be selected carefully. Suitable families have multiple affected and unaffected individuals from two or more generations available for analysis. 49 Families in which various members develop a rare form of cancer, such as sarcoma, are more likely to have a mutation segregating in an inherited cancer gene compared to families affected by more common types of cancer, for example, adenocarcinomas of the lung, breast, prostate, and colon. 49 Therefore ideal families for genetic studies of familial clustering of cancers are those with multiple generations of affected and unaffected individuals and families with multiple cases of a rare form of cancer such as sarcoma. 1.8 Known cancer predisposition genes Over the last 30 years, more than 100 cancer predisposition genes have been identified using a variety of strategies. 134, Figure 1.1 shows the location of known cancer predisposition genes and a full list of known cancer predisposition gene is available in Appendix F. However, only a small proportion of familial 38, 162 cancer risk can be explained by established cancer predisposition genes. The use of family-based NGS strategies in this field may facilitate the discovery of rare genetic mutations that explain the remaining genetic risk for cancer predisposition if much of the missing genetic control is due to gene variants that are too rare to be picked up by GWA studies and have relatively large effects on risk. 14

46 1.9 Summary There have been a substantial number of studies performed to identify genetic risk variants associated with cancer. Linkage studies have identified high penetrant risk alleles associated with Mendelian autosomal dominant cancer predisposition syndromes. Association studies have been used to successfully identify lower penetrant variants associated with more common types of cancer. However, much of the heritability of cancer remains unexplained. The introduction of NGS technology has allowed the identification of rare variants that are expected to explain some of the missing heritability of cancer. Study considerations for using NGS in cancer research include sequencing both tumour and germline DNA to facilitate the differentiation of somatic and germline mutations and to use a family-based study design with multiple generations of affected and unaffected individuals and families with multiple cases of a rare form of cancer such as sarcoma. To date, there have been few studies on shared genetic risk factors in cancer cluster families that are not defined by a known familial cancer predisposition syndrome. This study will employ the approach of performing WES in cancer cluster families of mixed cancer types. WES will be conducted on both affected and unaffected individuals from cancer cluster families that have been identified by a sarcoma proband to identify rare cancer predisposing variants. Only one previous study has used NGS technology to investigate shared genetic risk variants across multiple cancer types. 147 This study will be the second WES study performed on cancer cluster families to identify shared genetic risk variants, and the first WES study to select cancer cluster families by a sarcoma proband. 15

47 1 p36.22 p34.3 p32.1 p22.3 p13.2 q21.1 q23.2 q31.1 q41 q43 2 p25.2 p23.1 p16.1 p11.2 q12.3 q21.3 q24.2 q32.2 q35 3 p25.3 p22.2 p21.1 p11.2 q13.2 q22.3 q26.1 q28 4 p16.1 p15.1 q12 q21.3 q25 q31.1 q p15.31 p13.2 q12.1 q14.2 q22.2 q31.2 q34 6 p24.3 p21.32 p11.1 q14.3 q22.1 q p22.1 p14.2 p11.1 q21.12 q31.1 q33 8 p23.1 p11.23 q12.2 q21.2 q p23 p13.2 q13 q22.2 q p15.1 p11.23 q11.23 q23.1 q p15.3 p13 q12.3 q14.1 q p13.31 p11.1 q13.3 q21.33 q p12 q12.2 q14.3 q p12 q11.2 q22.1 q p12 q12 q21.1 q p13.2 p11.1 q21 17 p13.2 q11.2 q p11.23 q12.1 q p13.2 q11 20 p12.3 q p12 q p12 q11.23 X p22.32 p21.2 q11.1 q21.31 q24 q27.3 Y p11.2 q Indicates position of known cancer predisposition gene Figure 1.1: Location of known cancer predisposition genes 16

48 1.10 Aims The identification of genes that predispose individuals to cancer is a high priority in human medical research. It is anticipated that this knowledge will drive a new era of personalised human medicine, potentially allowing tailoring of specific drug treatments and interventions. The use of NGS in families currently represents an optimal study design for the identification of rare genetic variants involved in the risk of cancer and other common complex human diseases. Waves of novel genetic discoveries using this approach are now regularly appearing in the literature. While it is sometimes difficult to distinguish sporadic from hereditary cancer, rare cancer, such as sarcoma, occurring twice within the one family is epidemiologically striking. 163 The identification of genetic risk factors for cancer will be a significant contribution to medicine and particularly in the provision of health care to cancer patients and their families. The aims of this study are: 1. To perform WES on three cancer cluster families identified by a sarcoma proband using peripheral blood samples. 2. To identify candidate germline risk variants by prioritising and filtering structural and regulatory variants that segregate with cancer or sarcoma in the three families. 3. To perform a matched tumour and germline analysis on two myxoid liposarcoma patients using peripheral blood genomic DNA and genomic DNA isolated from sarcoma tumour tissue to distinguish somatic mutations. 4. To validate the most significant putative germline and somatic cancer predisposing mutations in unrelated sarcoma cases and cancer-free controls. 17

49 18

50 Chapter 2 Aim 1: Whole exome sequencing of three cancer cluster families identified by a sarcoma proband 2.1 Introduction Next Generation Sequencing (NGS) has provided tremendous insight into the genomic landscape of several tumour types, including defining tumour subtypes, identifying new druggable targets and understanding into the heterogeneity of many tumours. 164, 165 Protein-coding genes constitute approximately 1% of the human genome but harbour nearly 85% of the disease-causing mutations of 140, Mendelian diseases, although this may be due to ascertainment bias. Genetic variations discovered in coding regions of genes may inform immediate treatment choices and also further other therapeutic discoveries. 170, 171 Therefore, exome sequencing is an efficient approach for identifying actionable variants. The first aim of this study was to perform whole exome sequencing (WES) in three cancer cluster families ascertained from an index sarcoma patient. 19

51 2.1.1 Ion Proton platform The Ion Proton platform from Thermo Fisher Scientific is a benchtop semiconductor-based sequencing system for the human genome, exome or transcriptome sequencing. Semiconductor sequencing is based on the detection of hydrogen ions that are released during the polymerisation of DNA using a sequencing-by-synthesis approach. 172 The Ion Proton sequencing chemistry uses native deoxynucleotides (dntps) and electronic sensors to detect the release of hydrogen atoms as the dntps are incorporated into the growing DNA strand. 173 Microwells are sequentially flooded with each dntp to distinguish the order of each nucleotide. 173 Homopolymer runs are detected by the magnitude of the ph change to determine how many nucleotides were added. 173 Errors on the Ion Proton are mostly due to insertions and deletions in homopolymer runs due to the difficulty in evaluating the magnitude of signal when several dntps are incorporated in one cycle. 174 Automated sequencing analysis occurs using the Torrent Suite software that is preinstalled on the Torrent Server. The web-based interface can be used to plan, monitor and view the results of sequencing runs. The Torrent Suite base calling algorithm converts the raw file information into a sequence of bases and writes the sequence to an unaligned Binary Alignment/Map (*.bam) file. The *.bam file is then aligned using Torrent Mapping Alignment Program (TMAP). Variants are called using the Torrent Variant Caller (TVC). Both TMAP and TVC were developed specifically for Ion Torrent data and were used in this chapter. 20

52 2.2 Methods Families selected for whole exome sequencing The patients were recruited from the International Sarcoma Kindred Study (ISKS). The ISKS was initiated in 2008 to investigate the prevalence and nature of heritable risk in sarcoma populations. 175 The ISKS is a global genetic, biological, epidemiological, and clinical resource for researchers to investigate the hereditary characteristics of sarcoma. Patients were recruited from several sites across Australia, France, New Zealand, India, the United States of America, the United Kingdom, and Canada. The ISKS Steering Committee granted access to the database for this study under an ethically approved protocol (the University of Western Australia (UWA) Human Research Ethics Committee RA/4/1/6434). Patients with sarcoma (probands) were recruited from major sarcoma treatment centres, regardless of their family history of cancer. Individuals with adult-onset sarcoma (> 15 years old) were eligible for the ISKS. Family members were also invited to participate if the patient with sarcoma was < 45 years of age, or there was a significant family history of cancer. 175 Study questionnaires containing demographic, medical, epidemiological and psychosocial information were completed, including personal history of cancer or exposure to known risk factors for sarcoma. 176 Patients were also asked to donate a venous blood sample and tumour sample, as well as provide access to medical information and access to information about deceased relatives (collected from cancer registries and other health organisations). Medical history and treatment records were obtained for each proband where possible. 176 All reported cancer diagnoses were independently verified by medical records, Australian and New Zealand cancer registries or death certificates. There are now more than 1,300 families enrolled in the ISKS with detailed pedigree information and cancer incidence verified for each. More than 1,800 blood samples have been collected and approximately 2,100 questionnaires completed. The average age at onset for sarcoma in the ISKS cohort is 46.6 years (range 3-95 years) with the majority being sarcomas of soft tissue. Family members have reported over 2,000 other cancers. The average age at diagnosis for these other cancers is 57.9 years compared to 65.6 years in the general population

53 Since the establishment of the ISKS, several studies have focused on identifying TP53 germline mutations in Li-Fraumeni and the less stringent Li-Fraumeni-like syndrome in the cohort. 163, 176, 177 A previous study found pathogenic TP53 mutations in blood DNA of 20 of 559 sarcoma probands (3.6%) in the ISKS cohort. 176 The study of familial cancer cluster patterns in the ISKS identified 14% of the ISKS families with patterns of familial clustering without conforming to any known syndrome. 163 A more recent study using the ISKS discovered that more than half of the sarcoma patients had an excess of putatively pathogenic monogenic and polygenic germline variation in known and novel cancer genes using a case-control rare variant burden test. 178 The combination of findings that 14% of cancer cluster families in the ISKS do not conform to known syndromes and the excess of rare monogenic and polygenic germline mutations in more than half of the ISKS patients indicate the potential utility of this cohort to identify novel genetic risk factors for sarcomas and cancer clustering in families. Three ISKS families that do not conform to known cancer syndromes were targeted for selection in the current study and represented a unique opportunity to identify novel variants that may influence sarcoma or cancer development. These three families were selected for the current study based on the following selection criteria: The sarcoma proband must have blood and tumour biospecimens available The pedigree must contain a first degree relative with cancer also with germline samples available The pedigree must contain at least one unaffected relative with germline material available, and The family is not defined by TP53 or other known familial cancer susceptibility genes Family 1 (Figure 2.1) depicts a proband (Patient 1-III-1) who developed Ewing s sarcoma at 15 years of age, as well as a non-identical twin brother (Patient 1-III-2) who has not developed sarcoma. The proband s father (Patient 1-II-2) 22

54 developed myxoid liposarcoma at 39 years of age. Germline DNA was available from the proband and father, and from the proband s twin brother, mother (Patient 1-II-3), an aunt (Patient 1-II-1) and grandparents (Patient 1-I-1 and Patient 1-I-2), who were all unaffected by cancer. Family 2 (Figure 2.2) was identified by a proband (Patient 2-II-2) who developed myxoid liposarcoma at 61 years of age. The proband s father (Patient 2-I-1) developed prostate cancer at 71 years old, and two of the proband s sisters were diagnosed with skin melanomas at 44 (Patient 2-II-3) and 46 (Patient 2-II-2) years of age. Germline DNA was available for the proband, one of his unaffected children (Patient 2-III-1), three of his sisters (including an unaffected sister, Patient 2-II-4), and his parents (Patient 2-I-1 and Patient 2-1-2). In family 3 (Figure 2.3), there are two individuals with sarcoma; the proband (Patient 3-III-1) who developed a primitive neuroectodermal tumour (PNET) at 22 years of age, and her grandmother (Patient 3-I-1) who developed malignant peripheral nerve sheath tumour (MPNST) at 79 years old. The proband s father (Patient 3-II-1) was diagnosed with prostate cancer at 51 years of age, and the proband s aunt developed breast cancer at age 36. Germline DNA was available from the proband, her parents (Patient 3-II-1 and Patient 3-II-2), her unaffected brother (Patient 3-III-2), and her grandmother. 1-I-1 1-I-2 Key Affected male Affected female Unaffected male 1-II-1 1-II-2 Sarcoma (39) 1-II-3 Unaffected female Proband 1-III-1 Sarcoma (15) 1-III-2 Figure 2.1: Pedigree of family 1 23

55 2-I-1 2-I-2 Prostate (71) Key Affected male Affected female Unaffected male 2-II-1 Sarcoma (61) 2-II-2 Melanoma (46) 2-II-3 2-II-4 Melanoma (44) Unaffected female Proband 2-III-1 Figure 2.2: Pedigree of family 2 3-I-1 Sarcoma (79) Key Affected male 3-II-1 Prostate (51) 3-II-2 Affected female Unaffected male Unaffected female 3-III-1 Sarcoma (22) 3-III-2 Proband Figure 2.3: Pedigree of family 3 24

56 2.2.2 DNA extraction DNA extraction was performed by researchers at the Peter MacCallum Cancer Centre in Melbourne, Australia. Anti-coagulated blood was processed using a Ficoll gradient. DNA was extracted from the nucleated cell product using QIAamp DNA blood kit (Qiagen) Whole exome sequencing WES was performed by the candidate at the Curtin University - UWA Centre for Genetic Origins of Health and Disease (GOHaD). Two germline samples from Patient 3-I-1 and Patient 3-III-2 were badly degraded and of poor quality. Therefore, whole genome amplification was performed on these samples using a Qiagen REPLI-g Mini Kit (Qiagen) as per the manufacturer s instructions. Exome library preparation was performed using the Thermo Fisher Scientific Ion AmpliSeq Exome RDY Kit as per the manufacturer s instructions. Libraries were loaded onto the Ion P1 v2 BC Chip (Thermo Fisher Scientific) using the Ion Chef and sequenced on the Ion Proton as per the manufacturer s instructions. An overview of the WES pipeline is shown in Figure Library preparation The target regions were amplified using the Ion Ampliseq Exome RDY Library Preparation from 100 ng of genomic DNA in the Ion Ampliseq Exome RDY plates and the Ion Ampliseq HiFi Mix. The amplicons were treated with FuPa reagent to digest the primers partially and to phosphorylate the amplicons. The amplicons were then ligated to Ion Xpress Barcode Adapters, purified and dissolved in 50 µl of Low TE. Validation of enrichment and quantification of target DNA were performed on the ViiA 7 (Thermo Fisher Scientific). Three 10-fold dilutions of Escherichia coli control library were prepared at 6.8 pm, 0.68 pm and pm. 9 µl of each control library and each sample were added to wells of a 96-well qpcr plate as well as 11 µl of the reaction mixture for a total reaction volume of 20 µl. The qpcr was run for 40 cycles. 25

57 Samples 19 germline samples Library Preparation Ion Ampliseq Exome RDY Library Preparation Sequencing platform Life Technologies Ion Proton Quality check Torrent Suite software Sequence alignment Torrent Mapping Alignment program Variant calling Torrent Variant Caller plugin Genome Analysis Toolkit UnifiedGenotyper Merge using bcftools BCFtools intersect Figure 2.4: Whole exome sequencing pipeline flowchart 26

58 Exome sequencing Run plans were created for each chip with the barcode and sample identity number on the Torrent Browser server. The plans were created using the Torrent Suite Software with the run parameters listed in Table 2.1. Table 2.1: Parameters used to create whole exome sequencing run plans using Torrent Suite software Parameter Application Kit Library kit type Template kit Specified DNA Ion Ampliseq Exome Kit Ion Ampliseq Exome RDY IC Kit 1x8 Ion Chef, Ion PI IC 200 kit Flows 520 Chip type Barcode set Reference library Plug ins Ion PI chip IonXpress Human genome build 19 (hg19) variantcaller and coverageanalysis The sample libraries were diluted to approximately 50 pm, the optimal input concentration. The Ion PI v2 BC chips were prepared for loading by performing alternate washes with 100% isopropanol, Ion PI Chip Preparation Solution, nuclease-free water, 0.1 M NaOH, and 1X Ion Chip Priming Solution as per the manufacturer s instructions. The Ion PI IC Reagents 200 cartridge was removed from the freezer and warmed to room temperature 45 min before the Ion Chef Instrument run. The Ion Chef Instrument was loaded with treated Ion chips, consumables, reagents and libraries as per the manufacturer s instructions (Thermo Fisher Scientific). The Ion Chef Instrument run completed overnight. 27

59 The following day, the Ion Proton Sequencer was initialised as per the manufacturer s instructions (Thermo Fisher Scientific). The Ion chips were unloaded from the Ion Chef Instrument, and the first chip was loaded into the Ion Proton Sequencer. The second chip was stored in a container at 4 C until 20 min before the end of the first run. When the first run was completed, the second chip was loaded immediately for sequencing Sequence alignment and variant calling The Torrent Suite software (Life Technologies, v ) Torrent Variant Caller (TVC) was used to perform base calling. The resulting base calls were stored in an unmapped *.bam format. The Torrent Suite Torrent Mapping Alignment Program (TMAP) was used to align sequencing reads to the reference genome using human genome build 19 (hg19). Some or all of the reads produced by the WES pipeline are used as input for TMAP, along with the reference genome and index files. The output from TMAP is a mapped *.bam file Variation to sequence alignment and variant calling Torrent variant caller plugin As an additional measure, base calling was performed a second time using the TVC Plugin (Life Technologies, version 5.0.0). The TVC Plugin software was installed on Magnus (Pawsey Centre), a Cray XC40 supercomputer. The AmpliSeq Exome capture browser extensible data (*.bed) file from Life Technologies was used as the target region *.bed and primer trim *.bed file (available from The output is a variant call format (*.vcf) file containing meta-information lines, a header line and data lines for each position in the genome. 179 Each individual was called separately using TVC, generating 19 individual *.vcf files. The details used to run the TVC Plugin on Magnus are outlined in Table

60 Table 2.2: Parameters used to run the Torrent Variant Caller plugin to call bases Parameter Input bam Reference fasta Region bed Primer trim bed Error motifs Specified All *.bam files from the Ion Proton hg19.fasta AmpliSeqExome designed.bed AmpliSeqExome designed.bed ampliseqexome_germline_p1_hiq_motifset.txt Each of the 19 patients was called individually and then merged using Binary Variant Call Format Tools (BCFtools) vcf-merge 179 to create a single *.vcf file. As TVC only calls individual *.bam files, there is uncertainty whether a position is truly missing or is reference homozygous. BCFtools missing-to-reference 179 was also run on the merged file to fill unknown positions to homozygous reference (0/0) Genome analysis toolkit The Genome Analysis Toolkit (GATK, version 3.4.0) UnifiedGenotyper 180 was used in addition to the single sample calling to sort, index and call the *.bam files to ensure base calling accuracy. GATK can perform multi-sample calling. Therefore, all 19 patients were called together. GATK UnifiedGenotyper was used on a secure Linux server owned by GOHaD (operating system: Bio-Linux (based on Ubuntu )). UnifiedGenotyper uses a Bayesian genotype likelihood model to estimate the most likely genotypes and allele frequency in a population of samples simultaneously and produces a genotype for each site. First, each sample was sorted using SAMtools sort and indexed using SAMtools index. 181 Picard CreateSequenceDictionary (version 2.4.1, was used to create a sequence dictionary for a reference sequence and then Picard BedToIntervalList was used to convert a *.bed file to Picard interval list format. The specifications used to run GATK UnifiedGenotyper on the server are outlined in Table

61 Table 2.3: Parameters used for Genome Analysis Toolkit UnifiedGenotyper to call bases Parameter Reference fasta Genotype likelihoods model Input bam Target interval list Out mode Metrics Specified hg19.fasta SNP All sorted *.bam files from the Ion Proton AmpliSeqExome bed EMIT_ALL_CONFIDENT_SITES Directory for metrics Stand-conf-call 50.0 Stand-emit-conf 10.0 Annotation AlleleBalance Intersect variant calls from Torrent Variant Caller and Genome Analysis Toolkit The resulting *.vcf files from both TVC and GATK were combined using BCFtools intersect (isec) 181 exact allele match to identify the common calls between TVC and GATK. This tool created both intersections and complements of the TVC and GATK *.vcf files. The intersect data from both callers was used for the remainder of the analysis Recalibrate variants GATK VariantRecalibrator 180 was used to assign a well-calibrated probability to each variant call in a call set. This tool has a two stage process called Variant Quality Score Recalibration (VQSR). The first pass is performed by VariantRecalibrator 180 and consists of creating a Gaussian mixture model by looking at the distribution of annotation values over a high quality subset of the input call set and then scoring all input variants according to the model. 180 The recalibrated variant quality score provides a continuous estimate of the probability that each variant is correct, allowing one to partition the call sets into quality tranches. 182 The 30

62 primary purpose of the tranches is to establish thresholds within the data that correspond to particular levels of sensitivity relative to the truth sets. The second pass is performed by the ApplyRecalibration tool 180 that applies the model parameters to each variant in input *.vcf files to produce a recalibrated VCF file in which each variant is annotated with its variant quality score log-odds (VQSLOD) value. 182 This step also filters the calls based on this new logarithm of the odds (LOD) score by adding Pass for variants that meet the specified threshold, and LowQual in the FILTER column for variants that do not meet the specified LOD threshold. 180 The filter level selected for the ApplyRecalibration tool was Genotype concordance Concordance was measured in three patients that had previously been genotyped to validate the genotype calls. The three patients, Patient 1-II-2, Patient 2-II-1 and Patient 3-III-1, all sarcoma cases, had been genotyped previously through the ISKS using an Agilent HaloPlex custom panel of gene coding sequence capture. Genotype calls were compared across the three sarcoma cases and to determine how many calls (either 0/0, 0/1 or 1/1) were the same between the intersect file and previous genotyping using the Agilent HaloPlex custom panel. Any discordant variants were checked in the *.vcf files. The *.bam files were 183, 184 also visually examined in Integrative Genomics Viewer (IGV, version ). 2.3 Results Families selected for whole exome sequencing This study included 19 patients from three multigenerational mixed cancer families. Of these, 11 (58%) were female, and nine (47%) had been diagnosed with cancer. The average age of the patients at the time of blood collection was 55.3 years (range: 15 years to 90 years) and the average age of cancer (including sarcoma) onset was 47.5 years (range: 15 years to 79 years). The average age of onset in the three families is younger than the average age of onset of all 31

63 cancers in the whole ISKS cohort (57.9 years) but similar to the age of onset of sarcomas (46.6 years) Whole exome sequencing Table 2.4 shows the summary statistics generated by the Torrent Suite software. The average depth of coverage across all samples was reads, which is a sufficient depth for detecting single nucleotide variants (SNVs). 185, 186 The average number of mapped reads was 38,484,361, and the average total genotyping rate was 98.9%. Table 2.4: Depth of coverage summary from Torrent Suite Patient Mapped reads On target Mean Depth Number of variants 1-I-1 43,848, % ,625 1-I-2 28,509, % ,690 1-II-1 28,343, % ,334 1-II-2 38,178, % ,113 1-II-3 39,158, % ,915 1-III-1 37,229, % ,670 1-III-2 42,568, % ,641 2-I-1 33,480, % ,574 2-I-2 48,585, % ,220 2-II-1 35,464, % ,678 2-II-2 45,333, % ,491 2-II-3 46,884, % ,238 2-II-4 36,173, % ,517 2-III-1 30,353, % ,282 3-I-1 34,870, % ,493 3-II-1 42,063, % ,329 3-II-2 40,663, % ,846 3-III-1 47,344, % ,337 3-III-2 32,146, % ,169 Average 38,484, % ,482 32

64 2.3.3 Variant calling 5,099,324 unknown positions were changed to reference positions in the merged TVC *.vcf files using BCFtools missing-to-reference. In total, 109,503 variants were called by TVC and 238,530 variants were called by GATK UnifiedGenotyper. Figure 2.5 shows a diagram of the number of calls by TVC and GATK and the intersection of both callers. The intersect file from both callers contained 94,263 variants for all 19 patients. 144,267 94,263 15,240 Genome Analysis Toolkit Intersect Torrent Variant Caller Figure 2.5: The number of variants called by Torrent Variant Caller and Genome Analysis Toolkit UnifiedGenotyper, and the number of variants that were called by both callers (intersect) Recalibrate variants Figure 2.6 shows the tranche plot generated by GATK VariantRecalibrator. The first tranche (90), has the lowest value of truth sensitivity but the highest value of novel Ti/Tv, is very specific but less sensitive. 187 Each subsequent tranche introduces additional true positive calls along with a growing number of false positive calls. 187 Table 2.5 shows the 99.0 tranche used in this study that has 85,941 known calls and 3,097 novel calls with 49,447 accessible truth sites. In total, 48,952 calls were made in tranche The resulting file now has a new column generated by VariantRecalibrator that has pass or low quality for each variant. 33

65 Figure 2.6: Genome Analysis Toolkit VariantRecalibrator tranche plot X-axis: the number of novel variants called. Y-axis: the novel transition to transversion ratio and the overall truth sensitivity. TP (true positive): exact match of non-reference genotype. FP (false positive): additional alternate allele in WES genotype. Table 2.5: Genome Analysis Toolkit VariantRecalibrator tranche results Tranch minvqslod Known Novel Truth sites Called ,136 at ,254 at ,447 accessible 44, ,941 at ,097 at ,447 accessible 48, ,789 at ,200 at ,447 accessible 49, ,975 at ,528 at ,447 accessible 49,447 34

66 Figure 2.7 shows the 2D projection of mapping quality rank sum (MQRankSum) test versus haplotype score by marginalising over the other annotation dimensions in the model. The mapping quality rank sum test is the u-based z-approximation from the Mann-Whitney Rank Sum Test 188 for mapping qualities, that is, reads with reference bases versus those with the alternate allele. 187 This measure can be used to evaluate the likelihood of SNPs being real. Figure 2.7: Genome Analysis Toolkit VariantRecalibrator projection for mapping quality rank sum (MQRankSum) versus haplotype score The upper left panel shows the probability density function that was fitted to the data. Green: high quality. Red: lowest quality. The remaining three panels give scatter plots in which each single nucleotide polymorphism (SNP) is plotted in the two annotation dimensions (MQRankSum and HaplotypeScore) in a point cloud. In the upper right panel, SNPs are coloured black and red to show which SNPs are retained and filtered, respectively, by applying the variant quality score recalibration procedure. The lower left panel colours SNPs green, grey, and purple to give a sense of the distribution of the variants used to train the model. Green SNPs: found in the training sets. Purple: given the lowest probability of being true. The lower right panel colours each SNP by their known/novel status. Blue: known SNPs. Red: novel SNPs. 35

67 2.3.5 Genotype concordance A total of 212 positions across three previously genotyped individuals were used to compare genotype calls from WES and Agilent HaloPlex custom panel (Figure 2.8). Of those 212 positions, 77 were not called in the WES data due to low coverage or position of the primers. Of the remaining 135 positions, 123 calls (91%) were concordant between the two data types and 12 calls (9%) were discordant. Of the 12 discordant calls, two of the calls were in Patient 3-III-1 and were called at 1/1 using the Agilent HaloPlex custom panel data and called as 0/1 in the WES data. The remaining ten discordant calls were all in Patient 2-II-1 and were called as 0/0 from the Agilent HaloPlex custom panel data and either 0/1 (6 calls) or 1/1 (4 calls) using the WES data. Both concordant and discordant calls were kept in the intersect file. The genotyping positions were all located in easy to map regions of the genome and may not reflect the true false positive to false negative rate for all positions. 36

68 Called variant by Agilent HaloPlex custom panel genotyping Concordant Called variant by Ion Proton whole exome sequencing Not called in whole exome sequencing data 77 Concordant 123 Discordant 12 Called as variant by Agilent HaloPlex 2 Called as variant by Ion Proton 10 TOTAL 212 Figure 2.8: Concordance of genotype calls between the Agilent HaloPlex custom panel and whole exome sequencing on Ion Proton for three patients Blue: Called homozygous alternate (1/1) by Agilent HaloPlex custom panel but called heterozygous (0/1) by Ion Proton whole exome sequencing. Green: called variant (0/1 or 1/1) by Ion Proton whole exome but called homozygous reference (0/0) by sequencing Agilent HaloPlex custom panel. 37

69 Table 2.6 shows the ten positions in Patient 2-II-1 in which the genotype calls are discordant between the Agilent HaloPlex custom panel and the WES genotype, that is, where the variant is called 0/1 in the intersect file but no variant is called by the Agilent HaloPlex custom panel. The genotype calls for Patient 2-II-1 were checked in the TVC *.vcf, the GATK *.vcf and the intersect file. The genotype calls for the ten positions were the same across the three files. The genotype results from WES for both parents of Patient 2-II-1 (Patient 2-I-1 and Patient 2-I-2) are included in the last two columns of Table 2.6. These results indicate the WES genotype calls for Patient 2-II-1 at these positions are likely correct, given the genotypes of both parents. Table 2.7 shows the two discordant variants for Patient 3-III-1 which were both called as homozygous alternate using the Agilent HaloPlex custom panel but called as heterozygous in the intersect file. The genotype calls for these two positions were checked in the TVC *.vcf file, the GATK *.vcf file and the intersect file. TVC called the first variant (chromosome 7) as 1/1 whereas GATK called the variant 0/1. Therefore the position is called as 0/1 in the intersect file. TVC called the second variant (chromosome 13) also as 1/1, GATK called the variant 1/1, however, in the intersect file the variant is called 0/1. For both variants, the parents of 3-III-1 (last two columns) have a homozygous alternate genotype call. On visual inspection of the *.bam files in IGV, Patient 3-III-1 appears to be also homozygous for the alternate allele at these positions. Therefore it appears the errors for these variant calls occurred when intersecting the *.vcf files. 38

70 Table 2.6: Discordant genotype calls between the Agilent HaloPlex custom panel and whole exome sequencing for Patient 2-II-1 Chr Position Ref Alt Agilent HaloPlex GT Intersect file GT 2-I-1 (Father) 2-I-2 (Mother) C T 0/0 0/1 0/0 1/ T C 0/0 1/1 1/1 0/ A G 0/0 0/1 0/1 (low reads) 1/ A G 0/0 1/1 1/1 1/ A G 0/0 0/1 0/1 (low reads) 1/1 (low reads) T C 0/0 1/1 0/1 (low reads) 1/ G C 0/0 1/1 0/1 1/ A G 0/0 0/1 0/1 0/ G A 0/0 0/1 0/1 0/ C T 0/0 0/1 0/1 0/1 Chr: chromosome. Ref: reference allele. Alt: alternate allele. GT: genotype. Low reads: less than 10 reads at this position. Table 2.7: Discordant genotype calls between the Agilent HaloPlex custom panel and whole exome sequencing for Patient 3-III-1 Chr Position Ref Alt Agilent HaloPlex GT Intersect file GT 3-II-1 (Father) 3-II-2 (Mother) T C 1/1 0/1 1/1 1/ G C 1/1 0/1 1/1 1/1 Chr: chromosome. Ref: reference allele. Alt: alternate allele. GT: genotype.

71 2.4 Discussion Evaluation of families used in this study It has long been recognised that cancer has a familial component. Genetic studies were traditionally performed on sets of related individuals, including Mendel s study of inheritance patterns in pea plants from parents to offspring that propose the underlying mechanisms of inheritance. 189 Pedigree studies have been used successfully to identify genes influencing a broad range of monogenic, highly penetrant traits. 161 There are several reasons why family studies are used for gene discovery. Firstly, pedigrees are more likely to represent a more homogeneous and limited set of causal genes which enhance the statistical power for gene discovery. 190 Secondly, clinical characteristics that are shared among family members also reduce heterogeneity for analysis. 190 Thirdly, the analysis of phenotypes among family members is controlled to some extent for both genetic background and environmental exposures. 190 Therefore, the background genetic variation is also controlled to some extent. Finally, family data allow a deeper level of genotyping quality control than is possible in studies of unrelated individuals. 190 There are also disadvantages of using families in genetic research. It can be more costly to recruit entire pedigrees compared to unrelated individuals. 190 However, the analysis of disease/trait segregation in pedigrees with known genetic markers has proven to be a robust approach to gene discovery. The study of familial cancer predisposition syndromes characterised by sarcoma probands has resulted in valuable insight into cancer biology and genetic risk. For example, the study of Li-Fraumeni syndrome defined the roles of the tumour suppressor gene, TP53, in the development of cancer. Since germline mutations in the TP53 gene were first identified in Li-Fraumeni syndrome families, the gene has also been implicated in the sporadic form of most cancers. 51 It is now known that the TP53 gene has a role in the regulation of the cell cycle, DNA repair, apoptosis, cellular metabolism, and senescence. 191 These findings have had a significant impact on the clinical management of familial cancer predisposition syndromes and cancers in general

72 The ascertainment of cancer cluster families by a sarcoma proband has also been used to study incidence and distributions of cancers in relatives of sarcoma probands in families not defined by known syndromes These studies found an increased cancer risk in relatives of sarcoma probands, and suggest the presence of shared underlying genetic risk variants independent of known cancer predisposition syndromes. 195, 196 The families selected for investigation in the current study were in this category, i.e. they were not defined by a known cancer predisposition syndrome and therefore represent an opportunity to identify novel risk variants associated with both sarcoma and cancer risk. The ISKS families selected for WES in the current study include sarcoma, prostate cancer and melanoma cases. The occurrence of these cancers in families has been previously reported in familial cancer syndromes such as Li-Fraumeni syndrome 51, 52, 198, 199 and familial atypical multiple mole melanoma (FAMMM) syndrome (characterised by mutations in the CDKN2A gene), as well as other non-fammm syndrome families, also found to have mutations in the CDKN2A gene. 202, 204 However, the three families selected do not have mutations in the CDKN2A gene and therefore represent an opportunity to identify novel genetic variants that may lead to the development of these cancers within a family. The number and size of pedigrees vary widely in genetic studies of familial cancer. The number of relatives can range from two family members to extended pedigrees with > 30 individuals. 205, 206 The families used in this study are similar in size to the families studied by Roach et al. (2010) to discover the causative gene for Miller syndrome and Shi et al. (2014) to identify rare POT1 variants in familial 207, 208 cutaneous malignant melanoma The use of whole exome sequencing to identify disease causing variants WES has been a powerful approach for identifying genes that underlie Mendelian disorders and complex traits. 141, 144, 209, 210 To date, most genes discovered that underlie rare Mendelian disorders have genetic variation in protein coding sequences 166, 211 that are predicted to have functional consequences and be deleterious. 41

73 WES has also been a powerful and efficient approach for the discovery of genetic mutations in various cancers, identifying more than 50 novel tumour-predisposing genes (Appendix B). The identification of clinically actionable driver mutations through WES has enabled the development of precision oncology therapies Many of the genes that have been implicated in hereditary sarcomas play a significant role in the cellular response to DNA damage that has led to the development 216, 217 of DNA repair targeted therapies. WES has the advantage of increased coverage of regions of interest (exons) at lower cost and higher throughput compared with current whole genome sequencing (WGS). 148 WES was therefore chosen for this study as an appropriate, affordable 139, 210 and robust in-house method Limitations of whole exome sequencing A weakness of WES is that it largely ignores variants residing in non-coding and intergenic regions that can affect gene expression. 218 Non-coding DNA plays an important role in gene regulation and 3D chromatin folding 219 However, the effects of non-coding variants on gene expression are not yet completely understood. 220 The effects of regulatory variation may be more subtle and may be more important in common complex diseases such as cancer compared to Mendelian diseases. 221 The relevance of regulatory variation to cancer susceptibility in humans is unclear, but it is possible that polymorphisms in non-coding regions 221, 222 might have an important role. As the costs of WGS decrease and analytical tools such as Encyclopedia of DNA Elements (ENCODE) 223 become more adept at interpreting the effects of non-coding variants, WGS will become more widespread. The use of WGS studies to investigate genetic variants in cancer cluster families may lead to the discovery of mutations in regulatory elements that add to the pool of disease-associated variants. 224 Structural variations (defined as DNA sequence alterations other than SNVs including insertions, deletions, duplications, inversions and translocations) were not examined using WES in this chapter. There are many challenges in somatic structural variation detection inherent in the limitations of NGS technologies, the complexities of tumour samples and the difficulties in structural variant 42

74 reconstruction. 227 As WGS technologies improve, the use of paired-end reads, deeper coverage and longer sequence reads will facilitate the examination of somatic structural variants in cancer The Ion Proton sequencing platform The Ion Proton generally shows similar performance to other high-throughput sequencing platforms. 228, 229 The Ion Proton is also known to produce high quality data at a comparable average depth and read length in addition to a faster 172, 229, 230 turnaround time compared to the Illumina HiSeq. The average percent of reads on target produced in this study was 95.08%. The measurement of reads on target is represented by the ratio of the number of reads within a target region to the total number of bases output by the sequencer, expressed as a percentage. Off-target regions refer to those areas that are located 5 and 3 to target regions (upstream, downstream, untranslated regions and intronic). The percentage of on-target reads are dependent on the platform used as each platform uses different target choices, bait lengths, bait density and molecules used for capture Base calling software The TVC software was developed specifically to call Ion Proton sequencing data. However, it cannot produce multi-sample variant call files. The advantage of using multi-sample calling is to distinguish non-variant genotypes between 149, 231 homozygous reference genotype and missing genotype in cohort analysis. Multi-sample variant calling reduces the probability of calling random sequencing errors and increases the likelihood of calling alleles of low frequency or low coverage in a single sample. 149 Therefore, the sensitivity and accuracy of base calling are improved. 149 When calling the samples individually using TVC, many positions had to be filled to reference homozygous, and it was impossible to distinguish missing from homozygous reference positions. GATK UnifiedGenotyper can perform multi-sample calling and can, therefore, distinguish between missing and reference homozygous positions. However, GATK is not suited to Ion Proton data as the Ion Proton platform produces markedly different data to the Illumina 43

75 platform. 232 There were over twice the number of variants returned from GATK UnifiedGenotyper (238,530) compared to the number returned by TVC (109,503). Anecdotally, GATK does produce a higher number of false positives which may account for the difference in variants called (up to 10 times as many as reported on online bioinformatics forums). An intersect file of the calls made by TVC and GATK UnifiedGenotyper was created to reduce the number of false positives in the final call set and to overcome the problem of single sample calling by TVC and the platform differences by using GATK. Previous studies have recommended using multiple callers to generate a final call set. 233, 234 A simple way to combine call sets is to take the intersection or union of calls as final calls. 234 However, this was a very rigorous approach that reduced the number of variants from 109,503 called by TVC and 238,530 from GATK UnifiedGenotyper to just 94,263 in the intersect file. Therefore, some true variants may have been excluded as a result of using the intersect file. However, this may be the best approach for reducing the number of false positive calls Concordance In this study, the concordance rate of genotype calls for 135 positions from WES and the Agilent HaloPlex custom panel was 91%. The concordance rate falls into the range supported by previous literature on the concordance rates of panel versus sequencing data. A previous study by Motoike et al. (2014) aimed to validate SNV calls by exome analysis. They sequenced 12 independent genomes from Japanese patients using the Ion Proton semiconductor sequencer for whole exome sequencing (average depth 109). 235 Reads were aligned to hg19 using TMAP and genotype calling was performed on each sample using TVC. 235 Single nucleotide polymorphism (SNP) calls based on the Illumina Human Omni (version 2.5-8) SNP chip data were used as the reference. They analysed a total of 79,143 SNPs on the autosomes and found the concordance rate between the Omni and Ion Proton calls to be %. 235 These figures are comparable to results reported in a previous study

76 The intersect file described in this chapter was used in Aim 2 of this study to identify candidate risk variants. None of the discordant calls were removed from the intersect file. However, due to the findings of the concordance analysis, particularly the wrong call found in the intersect file but not either of the original *.vcf files from TVC or GATK, each variant detected in the analysis of this data was visually verified in the *.bam file using IGV. 45

77 46

78 Chapter 3 Aim 2: Identification of candidate germline risk variants in three cancer cluster families 3.1 Introduction Whole exome sequencing (WES) generates data on a large number of variants, most of which are not relevant to the disease of interest as they do not have a functional effect at the protein or systemic level. 236 The second aim of this study was to use the WES data described in Chapter 2 to identify candidate germline risk variants that segregate with cancer or sarcoma in three cancer cluster families. The analysis of WES data requires comprehensive computational approaches and strategies to identify candidate risk variants or genes for a disease of interest Despite advances in sequencing platform technology, reference data sets, software, and analysis pipelines, there is no gold standard for the filtering and prioritisation of variants. However, many guidelines, tools, and online resources have been developed to assist in the identification of functional variants from WES. 47

79 3.2 Bioinformatic strategies for variant filtering and prioritisation in whole exome sequencing Annotation As the sequencing of cancer genomes can reveal thousands of mutations, an essential step in the interpretation of WES data is the annotation of variants and their potential effects on genes and transcripts. 240 Variant annotation is the process of assigning functional information to DNA variants. At a basic level, annotations can be used to identify genes, transcripts and genomic regions, and at a higher level, also predict the impact of the variant on the protein product. There are over 80 bioinformatic tools available for genomic annotation, many of which are available as web-based applications. 241 Most tools focus on the annotation of single nucleotide variants (SNVs) as they are easily identified and analysed. 242 However, an increasing number of tools are being developed to annotate copy number alterations and other structural variations including 241, insertions, deletions, inversions and translocations. The most common form of annotation is the provision of links to public databases such as the National Center for Biotechnology Information (NCBI) Short Genetic Variations Database (dbsnp) or the 1000 Genomes Project. 251, 252 The functional prediction of variants can result from a simple sequence-based analysis, region-based analysis, or evaluation of the structural impact on proteins. 242 The choice of annotation tool is largely dependent on the desired selection of variant annotations. A widely used annotation tool to identify the functional consequence of sequence variation is Annotate Variation (ANNOVAR). 245 ANNOVAR predicts the functional effects of variants on genes, as well as performing genomic region-based annotation and comparison of variants to existing databases. 245 ANNOVAR incorporates scores based on evolutionary conservation and in silico prediction of functional consequences. 48

80 Annotation of non-coding regions A significant portion of the reads obtained in WES come from outside of the designed target region. 253 In a typical WES study, approximately 40-60% of the reads are off target, and all or most of these off-target reads are usually ignored Three main types of off-target reads are found in WES data: reads from introns and intergenic regions, reads from the mitochondrial genome and reads from viral genomes. 218 Although WES is not designed to identify regulatory variants in intronic and intergenic regions, off-target reads should not be discarded as many changes outside the coding regions may be responsible for disease phenotypes. 253 Annotation also plays an essential role in the interpretation of off-target variants. Regulome Database (RegulomeDB) can be used to guide the interpretation of regulatory variants in the human genome to identify potential regulatory changes based on experimental data sets from the Encyclopaedia of DNA Elements (ENCODE) and other sources. 257 RegulomeDB also includes computational predictions and manual annotations to identify putative regulatory potential and identify functional variants. 257 RegulomeDB uses a heuristic scoring system based on the functional consequence of the variant Variant class filtering Variant filtering can be carried out using annotations for the genomic location and the variant class. Annotations from ANNOVAR can be used to identify intronic variants, exonic variants, intergenic variants, 5 and 3 -untranslated region (UTR) variants, splicing site variants, and upstream or downstream variants. 245 For exonic variants, ANNOVAR scans annotated messenger ribonucleic acid (mrna) sequences to identify and report amino acid changes, as well as stop-gain or stop-loss mutations. 245 Exonic missense, nonsense, stop-loss, frameshift and splice site variants all have potential to affect protein function and are retained during this filtering process. 211, 239 RegulomeDB scores can also be used to filter variants that are more likely to lie in a functional location. 49

81 3.2.3 Population frequency filtering Population frequency is one of the primary criteria for predicting if a variant is likely to have a functional effect on the encoded protein. 258 Some rare nonsense variants might be expected to have a larger functional impact than a frequently occurring one. 211, 259 The Exome Aggregation Consortium (ExAC) database is the biggest catalogue of protein-coding genetic variation to date and is intended to be used as a general population resource to filter variants, including, for example, minor allele frequency (MAF). 260, 261 The ExAC database is the aggregation and analysis of high-quality exome DNA sequence data for 60,706 individuals of diverse ancestries. 261 The ExAC database is recommended due to the allele frequencies being calculated from considerably more samples compared to the Exome Variant Server and the 1000 Genomes Project. 252, 260 In disease studies, a commonly used starting point for filtering is to remove variants with a MAF > 1% Evolutionary conservation Genomic Evolutionary Rate Profiling (GERP) uses a comparative genomics approach to identify putatively functional sequences by comparing similarity across divergent species to identify sequences that have been maintained during evolution. 262 Pathogenic mutations tend to have a markedly higher conservation than benign variants. 263, 264 GERP uses maximum likelihood evolutionary rate estimation for position-specific scoring. 262 GERP scores range from a maximum of 6.18 to a below-zero minimum (-12.36). Positive scores represent a substitution deficit (expected for sites under selective constraint), while negative scores represent a substitution surplus Functional impact prediction In silico analysis of functional consequences of a variant on protein function and estimates of evolutionary conservation are often used for prioritisation in genetic discovery studies. Non-synonymous variants that lead to an amino acid change in the protein product are of particular interest as amino acid substitutions 50

82 account for approximately half of the known genetic variants responsible for human inherited disease. 265 Sorting Intolerant From Tolerant (SIFT) and Polymorphism Phenotyping-2 (PolyPhen-2) are commonly used tools that can predict if an amino acid substitution will have an effect on the protein function. 266, 267 SIFT uses sequence homology to predict whether an amino acid substitution will affect protein function and potentially alter phenotype. 266 A SIFT score 0.05 is predicted to be damaging, and a score > 0.05 is predicted to be tolerated. PolyPhen-2 predicts the possible impact of amino acid substitutions on the stability and function of human proteins using structural and comparative evolutionary considerations. 267 A PolyPhen-2 score between 0.0 and 0.15 is predicted to be benign, a score between 0.15 and 1.0 is predicted to be possibly damaging, and a score between 0.85 and 1.0 is more confidently predicted to be damaging. 267 An alternative strategy for filtering variants is based on a priori knowledge of the functional involvement of variants or genes. For example, association studies with candidate genes have been used to identify a number of risk genes for complex diseases. 268 A candidate gene study takes advantage of and is limited by knowledge of the phenotype, tissues, genes and proteins that are likely to be involved or have been previously implicated in the disease. 268, 269 Assessing candidate genes possessing functional variants in the context of existing biomedical knowledge and known biomolecular functions can be used to produce a manageable set of variants for further validation or exploration. 239 Several next generation sequencing (NGS) studies have identified rare variants associated with disease using a candidate gene approach In addition to variant filtering based on annotation and functional impact predictions, strong genetic support is also necessary for assigning possible causality to variants identified using WES. 239 Evidence of genetic association or familial segregation should be supplemented by functional and bioinformatics support. 51

83 3.2.6 Association analysis in families Association analysis in families can identify genes that influence complex human traits and provide protection against population stratification. 275 Variance components models are a way to assess the amount of variation in a dependent variable that is associated with one or more random-effects variables. 276 Variance components analysis is widely used in the genetic analysis of quantitative traits in family studies. 275 This approach is favoured because it can accommodate pedigrees of any size, it allows both linkage and association analysis, and tends to be more robust than competing approaches. 275 Sequential Oligogenic Linkage Analysis Routines (SOLAR) is a software that performs variance components analysis in pedigrees. 277 Almasy and Blangero (1998) 278 extended the strategy developed by Amos (1994) 279 for pedigree-based variance components analysis to estimate the genetic variance attributable to the region around a specific genetic marker using SOLAR. Maximum likelihood methods that take into account relationships among family members can be used to determine association in a polygenic model in SOLAR Familial segregation Segregation analysis is a general method for evaluating the transmission of a disease or trait within pedigrees. Segregation analysis can be used to prioritise and filter variants by assessing the co-segregation of candidate variants with disease status. 276 This analysis distinguishes variants that segregate with the disease of interest and are absent in unaffected family members. Segregation analysis can be applied to any pedigree structure and works with both qualitative and quantitative traits Outline of chapter This chapter describes the annotation, filtering, prioritisation and segregation analysis of WES data to identify putative germline risk variants that are associated with cancer or sarcoma in three cancer cluster families. WES data from Chapter 2 was annotated using ANNOVAR and RegulomeDB. Putative structural and regulatory variants were filtered using genomic location and variant class or 52

84 RegulomeDB score. Three different strategies were used to further prioritise rare private variants, known rare variants and candidate gene variants. Prioritised variants were tested for association with sarcoma and cancer using SOLAR. Significant variants were assessed for familial segregation with disease. 3.3 Methods Ascertainment bias correction The families selected for this study were ascertained from the International Sarcoma Kindred Study (ISKS), 175 as described previously in Chapter 2. A weighted covariate using a probability unit (probit) regression was created in R 281 (bias reduction in binomial-response generalised linear models (brglm) library, version 3.1.2) 281 to account for ascertainment bias in the sample. Probit regression assigns a weight to each based on their case status and can be used as a covariate in modelling Intersection The intersect file created from the variant call files from the Torrent Variant Caller (TVC, version 5.0.0), and Genome Analysis Toolkit (GATK, version 3.4.0) UnifiedGenotyper in Chapter 2 was used in these analyses. This file consists of 94,623 variants Annotation and filtration ANNOVAR (version 2015Jun16) 245 was used to annotate the intersect file using gene-based annotation. Using the ANNOVAR annotation, variants were filtered to include only putative structural variants. Variant filtering retained loci if they: (1) were exonic, (2) were predicted to be nonsynonymous or resulting in a stop gain or stop loss, (3) were predicted to be deleterious or probably damaging in SIFT and PolyPhen-2 and, (4) had a GERP score < 3. 53

85 All remaining variants that were not classified as putative structural variants were annotated using RegulomeDB. 257 Putative regulatory variants that had a RegulomeDB score of 1a, 1b, 1c, 1d, 1e, 1f, 2a, 2b or 2c were retained as these scores represent the highest confidence that a variant lies within a functional location. Table 3.1 shows the classification of scores from RegulomeDB. Known expression quantitative trait loci (eqtl) for genes are associated with expression and are most likely to result in a functional consequence. 257 Other subcategories with high confidence for regulatory variants are transcription factor (TF) binding, TF motifs, Deoxyribonuclease (DNase) footprints and DNase peaks. 257 Table 3.1: Classification of Regulome database scores Score 1a Supporting data eqtl + TF binding + matched TF motif + matched DNase Footprint + DNase peak 1b eqtl + TF binding + any motif + DNase Footprint + DNase peak 1c 1d 1e 1f 2a 2b 2c 3a 3b eqtl + TF binding + matched TF motif + DNase peak eqtl + TF binding + any motif + DNase peak eqtl + TF binding + matched TF motif eqtl + TF binding / DNase peak TF binding + matched TF motif + matched DNase Footprint + DNase peak TF binding + any motif + DNase Footprint + DNase peak TF binding + matched TF motif + DNase peak TF binding + any motif + DNase peak TF binding + matched TF motif 4 TF binding + DNase peak 5 TF binding or DNase peak 6 Other eqtl: Expression Quantitative Trait Loci. TF: Transcription Factor. DNase: Deoxyribonuclease. 54

86 False positive variants that arise due to misalignment, inaccuracies and biases in the reference sequence can be identified and provisionally excluded during a search for disease-causing variants. Fuentes Fajardo et al. (2012) analysed WES data from 118 individuals in 29 families to create a list of 2,157 genes that are candidates for provisional exclusion from exome analysis. 282 All filtered variants in this study were cross-referenced to the exclusion list by Fuentes Fajardo et al. (Available in the paper s Supplementary material: Table S7 gene exclusion list final ) to determine if any results found in polygenic regions should be excluded to reduce the risk of false positives Prioritisation strategies Prioritisation using a rare private variants strategy The first prioritisation strategy was applied to the filtered variants from the intersect file to identify rare private variants. Rare private variants are defined as those unique to individuals or families, and those that have not been previously annotated. 283 A major driving hypothesis behind WES of complex diseases is that multiple, rare variants in protein-coding genes contribute to the disease/trait of interest. 284 The focus on rare genetic variation is supported by studies that predict that numerous functional and deleterious variants segregate in the population at frequencies too low (0.5-5%) to detect by genome wide association (GWA) studies. 128 Investigators have successfully used this approach to identify rare private variants after removing known variants with a reference SNP identification (rs ID) from further consideration if they are found in the International Haplotype Project (HapMap), 285 the 1000 Genomes Project, 286 or dbsnp. 251 The variants from the intersect file were filtered to remove those that had been previously annotated to prioritise rare private variants in this study Prioritisation using a known rare variants strategy The second strategy was used to prioritise known rare variants from the filtered intersect file using a population database and MAF information. By filtering the data from WES for rare variants that have been documented in a large database such as ExAC, variants that occur at a low frequency in the population 55

87 that may be associated with cancer are more likely to be prioritised in these cancer cluster families. The full list of variants from the ExAC browser were downloaded (version 0.3.1, 30 August 2016). Variants from a complete list of ExAC browser variants with a MAF 0.01 (1%) and that were also in the intersect file were selected Prioritisation using a candidate gene strategy The prioritisation of candidate genes based on a priori knowledge of cancer biology was the third prioritisation strategy used on the filtered intersect file in this study. The variants from the intersect file were filtered to prioritise those detected in 119 known cancer and sarcoma genes including 25 kb upstream and downstream of the gene to include any potential regulatory variants captured in off-target reads. Candidate genes were selected from two cancer gene panels and a search of the Online Mendelian Inheritance in Man (OMIM) database. 287 Cancer genes were chosen from the HaloPlex Cancer Research Panel, 288 and Illumina s MiSeq and TruSeq Cancer Panels. 289 Both panels are NGS target enrichment panels that were designed for known cancer hotspots. The panels contain genes found in previous research to be associated with a broad range of cancer types as well as with published drug targets. Candidate genes from the results of a search of the OMIM database for genes known to be associated with the specific sarcoma subtypes in the three families were also included. 287 The full list of cancer genes used in the prioritisation process can be found in Appendix G. The variants present in both the intersect file and in the candidate genes were selected Methods for testing association of variants with cancer phenotypes SOLAR (version 7.6.4) 277 was employed to estimate and test the significance of association under a polygenic model for quantitative phenotypes (age at onset of cancer and age at onset of sarcoma) and disease status (cancer and sarcoma). Covariates included were age and sex of the participant, and the age sex interactions along with a weighting factor assigned to each individual to correct for the ascertainment 56

88 bias. Analysis of disease status as discrete binary traits was performed using a liability threshold model in SOLAR. This model employs probit regression for the mean effect component and a standard random effects variance component model for the residual additive genetic component of variance. 278, 290 As variance component models are highly influenced by kurtosis (a descriptor of the shape of a probability curve), the quantitative phenotypes were inverse normalised using the SOLAR function, inorm Bonferroni correction Bonferroni correction was performed on each annotated variant list to correct for multiple testing. 292 Corrections were performed for each method based on the number of variants in the prioritised list. Any significant variants after correcting for multiple testing, or nominal variants (p-value < 0.05), were investigated for co-segregation in the families Familial segregation analysis Three assumptions were used to determine familial segregation. First, the variant will be rare (shared only by cases in one family). Second, every carrier of a putative disease-causing variant will have the phenotype (complete penetrance). Third, every individual with the disorder will carry the putative disease-causing variant (100% probability of observing a genotype given the phenotype). 284 Due to the segregation analysis assumptions, it was hypothesised that variants identified by this approach would be private mutations that co-segregate with cancer or sarcoma in each family. The genotypes of any variants found to segregate with the phenotype of interest were visually confirmed by importing the Binary Alignment/Map (*.bam) files into Integrative Genomics Viewer (IGV, version 183, ) by determining the number of reads for each allele Evidence further supporting candidate risk genes The candidate germline risk variants and the genes in which they arise were further examined for association with cancer pathogenesis using several in silico resources including the Catalogue of Somatic Mutations in Cancer (COSMIC),

89 the pathway unification database (PathCards), 293 gene ontology (GO) annotations, 294 PubMeth (a database of methylation in cancer), 295 and NCBI. 296 A PubMed search was performed using a string ( gene name ) AND (cancer OR malignancy OR tumor* OR tumour* OR sarcoma) in April Abstracts were screened for relevance to the current study. 3.4 Results Variant prioritisation The intersect file containing 94,263 variants was annotated with ANNOVAR and RegulomeDB and variants in known polymorphic regions were removed. Approximately 42% of variants were exonic and 51% were intronic (Table 3.2). Less than 1% of variants were intergenic. Of the exonic variants, approximately 48% were nonsynonymous, and 51% were synonymous, with 0.5% classified as stop gain and loss variants. Table 3.2: Functional annotation of intersect file using ANNOVAR Function Percentage Exonic Nonsynonymous Synonymous Stop gain/loss 0.50 Unknown 1.35 Intronic Intergenic 0.04 Upstream/downstream 0.68 UTR 4.96 Other

90 Prioritisation using a rare private variants strategy The first prioritisation method was employed to identify rare, novel variants not previously reported in reference data sets. Of the 94,263 variants in the intersect file, 4,425 variants had not previously been annotated with an rs ID number. Of these, 1,858 (42%) were exonic variants and 1,184 (64%) were nonsynonymous Prioritisation using a known rare variants strategy The second prioritisation method was used to identify known rare variants using the ExAC public database. There were over 10 million variants in the ExAC browser (release 0.3.1, 30 March 2016). Of those 10 million variants, 3,686,062 variants had a MAF of less than Of the ~3.7 million rare variants, 8,840 variants were also in the intersect file. Of these, 5,184 (59%) were exonic and 2,815 (54%) were nonsynonymous Prioritisation using a candidate gene strategy The third prioritisation method was based on a priori knowledge of cancer and sarcoma. The results of the WES intersect file were filtered to only those variants in known cancer and sarcoma genes (1,297 variants). Of these variants, 806 were in the known cancer genes listed in Appendix G. The remaining 491 variants were located in regions upstream and downstream (25 kb) of each known cancer gene. Appendix H contains a table of variants in the upstream and downstream regions of the cancer genes that were also prioritised using this method. Of the 1,297 variants, 487 (38%) were exonic and 211 (43%) were nonsynonymous Summary of annotated variants from each prioritisation strategy A summary of the annotated variants from each prioritisation strategy is presented in Table 3.3. The first section of the table shows the number of variants prioritised by each strategy, followed by the genomic location of variants, exonic function and functional prediction. The final section of the table shows the number of variants classified as putative structural and functional variants. The results of each prioritisation strategy were tested for significant associations with cancer phenotypes using SOLAR. 59

91 60 Table 3.3: Summary of variant annotation using Annotate Variation and Regulome Database for each prioritisation strategy Strategy Rare private variants Known rare variants Candidate gene variants Number of variants prioritised 4,425 8,840 1,297 Location Exonic 1,858 5, Intronic 2,170 3, Downstream Upstream untranslated region untranslated region Splicing Non-coding RNA Intergenic Upstream/downstream /3 untranslated region Exonic function Nonsynonymous 1,184 2, Stop gain Stop loss Synonymous 601 2, Unknown

92 Strategy Rare private variants Known rare variants Candidate gene variants Functional prediction Deleterious in SIFT and PolyPhen Tolerated in SIFT and PolyPhen , Unknown in SIFT and PolyPhen-2 3, Regulome database score < Classification Putative structural variants Putative regulatory variants SIFT: Sorting Intolerant From Tolerant. PolyPhen-2: Polymorphism Phenotyping-2. 61

93 3.4.2 Rare private variants Association analysis in SOLAR The annotated rare private variants (Table 3.3) were tested for association with cancer phenotypes using SOLAR. The results from SOLAR were corrected for multiple testing using the Bonferroni method with the number of prioritised variants (4,425). The significance level after correction was α < 1.23 x No variants were significantly associated with a cancer phenotype after correcting for multiple testing. As the variants prioritised by this strategy were rare, novel variants, all nominally significant variants (p-value < 0.05) were visually confirmed using IGV to determine if they could be due to alignment or calling error. Any variants located near an insertion or deletion or on the edge of a gap or read block were removed. Table 3.4 contains a summary of the nominally significant variants (p-value < 0.05) for the age at onset of cancer, the age at onset of sarcoma, and cancer status. The results show eight variants nominally associated with age at onset of cancer, six variants nominally associated with age at onset of sarcoma, and two variants showing nominal association with cancer status. There were no variants with a p-value < 0.05 for sarcoma status. Of the total variants, eight were associated with a single cancer phenotype, and four variants were associated with more than one cancer phenotype. Two variants were associated with age at onset of cancer and cancer status, and two variants were associated with age at onset of cancer and age at onset of sarcoma. As these were rare variants without an rs ID, MAF data from the 1000 Genomes Project database and annotation using RegulomeDB was not available. Therefore, all the variants identified by this prioritisation strategy were rare risk alleles. 62

94 Table 3.4: Summary of SOLAR association results for rare private variants Chr:Pos Gene p-value Beta SE Exonic function SIFT PolyPhen-2 GERP Ref Alt MAF Age at onset of cancer 8: ARHGAP NS D D 5.37 C T : NPPC NS D D 5.29 C G : NELFCD NS D D 5.84 T C : DNAH NS D D 4.05 G A : LRRC16A NS D D 5.94 G A : FGD NS D D 4.29 C A : LTBP Unknown D D 3.77 C A : RIMS NS D D 5.65 G A 0.05 Age at onset of sarcoma 17: FADS6 < NS D D 5.15 A G : DNAH NS D D 4.05 G A : LRRC16A NS D D 5.94 G A : LRRC4B NS D D 3.45 C G : MON1B NS D D 3.61 G T : DHX NS D D 4.89 A G 0.18 Cancer status 8: ARHGAP NS D D 5.37 C T : LTBP Unknown D D 3.77 C A Chr:Pos: Chromosome:Position. SE: Standard Error. SIFT: Sorting Intolerant from Tolerant score. PolyPhen-2: Polymorphism Phenotyping-2. GERP: Genomic Evolutionary Rate Profiling score. Ref: reference allele. Alt: alternate allele. MAF: Minor Allele Frequency in the study population. NS: nonsynonymous. D: deleterious.

95 Segregation analysis results Of the 12 variants identified, seven were seen only in one family (ARHGAP39, NELFCD, LTBP4, RIMS1, DNAH9, LRRC16A and FADS6 ). However, using all three criteria for familial segregation, only one conserved deleterious variant in the ARHGAP39 gene showed nominal association with age at onset of cancer and cancer status and complete familial segregation in family 3 (Figure 3.1). Each family member with cancer was heterozygous at this position, whereas unaffected family members were homozygous for the reference allele at this position. None of the other prioritised rare private variants showed complete familial segregation in any of the families according to the familial segregation criteria. 3-I-1 Sarcoma Key Affected male 3-II-1 Prostate 3-II-2 Affected female Unaffected male Unaffected female Proband 3-III-1 Sarcoma 3-III-2 Patient Genotype at position in ARHGAP39 gene Read depth Ref, alt Patient 3-I-1 C/T 18,16 Patient 3-II-1 C/T 38,30 Patient 3-II-2 C/C 66,0 Patient 3-III-1 C/T 37,57 Patient 3-III-2 C/C 13,0 Figure 3.1: Genotypes for the ARHGAP39 variant that shows segregation in patients with cancer in family 3 64

96 3.4.3 Known rare variants Association analysis in SOLAR The annotated known rare variants (Table 3.3) were tested for association with cancer phenotypes using SOLAR. The results from SOLAR were corrected for multiple testing using the Bonferroni method with the number of variants prioritised (8,840). The significance level after correction was α < 5.66 x No variants were significant after correcting for multiple testing. Table 3.5 contains a summary of the nominally associated variants (p-value < 0.05) for the age at onset of cancer, the age at onset of sarcoma, and cancer status. The results include ten variants that showed nominal association with age at onset of cancer (eight putative structural and two putative regulatory variants), one putative regulatory variant that showed nominal association with age at onset of sarcoma, and 15 variants showing nominal association with cancer status (12 putative structural and three putative regulatory variants). There were no variants showing association with a p-value < 0.05 for sarcoma status. Of all the variants, 12 were associated with a single cancer phenotype, and seven variants were associated with more than one cancer phenotype. Of the latter, all seven variants were associated with both cancer status and age at onset of cancer. 65

97 66 Table 3.5: Summary of SOLAR association results for known rare variants Chr:Pos Gene p-value Beta SE Exonic function SIFT PolyPhen-2 RegulomeDB GERP Ref Alt MAF 1000G MAF Age at onset of cancer 1: ZFP69B NS D D 3a 3.33 C G : BEAN NS D C A : UVSSA NS D D G A : C16orf NS D D 2b 5.22 T C : C6orf NS D D A T : ADAMTS NS D D C T : DLG NS D D C G : MYC < NS T D 2b 3.91 A G : KIF2C S.. 2b. G A : ABCB S.. 2c. G A Age at onset of sarcoma 16: SF3B3 < Int.. 2b. T G Cancer status 16: C16orf NS D D 2b 5.22 T C : ZFP69B NS D D 3a 3.33 C G : UVSSA NS D D G A : ADAMTS NS D D C T : DLG NS D D C G

98 Chr:Pos Gene p-value Beta SE Exonic function SIFT PolyPhen-2 RegulomeDB GERP Ref Alt MAF 1000G MAF 13: STARD NS D D C T : HR NS D D G A : RRS NS D D C A : SYNPR NS D D C T : MOCS NS D D G A : SPATA NS D D A T : C6orf NS D D A T : ABCB S.. 2c. G A : IRAK NS T b C G : KRTAP NS T b T C Chr:Pos: Chromosome:Position. SE: Standard Error. SIFT: Sorting Intolerant from Tolerant score. PolyPhen-2: Polymorphism Phenotyping-2. GERP: Genomic Evolutionary Rate Profiling score. Ref: reference allele. Alt: alternate allele. MAF 1000G: Minor Allele Frequency in 1000 Genomes Project. MAF: Minor Allele Frequency in study population. NS: nonsynonymous. S: synonymous. Int: intronic. UTR3: 3 untranslated region. UTR5: 5 untranslated region. D: deleterious..: not annotated in database. 67

99 Segregation analysis results Of the 19 variants, 13 were only seen in one family (ZFP69B, BEAN1, UVSSA, C16orf96, ADAMTS14, DLG5, KIF2C, ABCB5, STARD13, HR, RRS1, SYNPR and MOCS2 ). Using the three criteria for familial segregation, six variants showed complete familial segregation. Two conserved, deleterious variants showed complete familial segregation in family 2 (Figure 3.2). An exonic nonsynonymous variant in the C16orf96 gene showed nominal association with age at onset of cancer and onset of cancer. A synonymous variant in the ABCB5 gene also showed nominal association with age at onset of cancer and cancer status. Each family member with cancer was heterozygous at these positions, whereas unaffected family members were homozygous for the reference allele at these positions. 2-I-1 2-I-2 Prostate Key Affected male Affected female Unaffected male 2-II-1 Sarcoma 2-II-2 Melanoma 2-II-3 Melanoma 2-II-4 Unaffected female 2-III-1 Patient Genotype at position in C16orf96 gene Read depth Ref, alt Proband Genotype at position in ABCB5 gene Patient 2-I-1 T/T 119,0 G/G 69,0 Patient 2-I-2 T/C 28,38 G/A 6,4 Read depth Ref, alt Patient 2-II-1 T/C 63,38 G/A 32,41 Patient 2-II-2 T/C 36,27 G/A 54,65 Patient 2-II-3 T/C G/A 41,38 Patient 2-II-4 T/T 62,1 G/G 65,0 Patient 2-III-1 T/T 42,0 G/G 96,0 Figure 3.2: Genotypes for the C16orf96 and ABCB5 variants that show segregation in patients with cancer in family 2 68

100 Using the three criteria for familial segregation, four conserved, deleterious variants showed complete familial segregation in family 3 (Figure 3.3). Exonic variants in the ZFP69B and UVSSA gene showed nominal association with both age at onset of cancer and cancer status in family 3. Two exonic variants in the BEAN1 and KIF2C genes showed nominal association with age at onset of cancer in family 3. All patients with cancer in family 3 were heterozygous at these positions, and unaffected family members were homozygous for the reference allele at these positions. None of the other prioritised known rare variants showed complete familial segregation in any of the families according to the familial segregation criteria. 69

101 70 3-II-1 Prostate 3-I-1 Sarcoma 3-II-2 Key Affected male Affected female Unaffected male Unaffected female 3-III-1 Sarcoma 3-III-2 Proband Patient Genotype at position in ZFP69B gene Read depth Ref, alt Genotype at position in UVSSA gene Read depth Ref, alt Genotype at position in BEAN1 gene Read depth Ref, alt Genotype at position in KIF2C gene Patient 3-I-1 C/G 27,19 G/A 58,31 C/A 28,26 G/A 59,52 Patient 3-II-1 C/G 29,37 G/A 75,69 C/A 18,19 G/A 50,66 Patient 3-II-2 C/C 118,0 G/G 129,0 C/C 62,0 G/G 126,0 Read depth Ref, alt Patient 3-III-1 C/G 24,41 G/A 127,64 C/A 44,30 G/A 125,107 Patient 3-III-2 C/C 44,0 G/G 85,0 C/C 52,0 G/G 118,0 Figure 3.3: Genotypes for the ZFP69B, BEAN1, UVSSA and KIF2C variants that show segregation in patients with cancer in family 3

102 3.4.4 Candidate gene variants Association analysis in SOLAR The annotated candidate gene variants (Table 3.3) were tested for association with cancer phenotypes using SOLAR. The results from SOLAR were corrected for multiple testing using the Bonferroni method with the number of variants prioritised (1,297). The significance level after correction was α < 3.86 x No variants were significant after correcting for multiple testing. Table 3.6 contains a summary of the nominally associated variants (p-value < 0.05) for the age at onset of cancer, the age at onset of sarcoma, and cancer status. The results include 14 variants that showed nominal association with age at onset of cancer (2 putative structural and 12 putative regulatory variants), two putative regulatory variants that showed nominal association with age at onset of sarcoma, and 12 variants that showed nominal association with cancer status (one putative structural and 11 putative regulatory variants). There were no variants showing an association with a p-value < 0.05 for sarcoma status. Of the total variants, 18 variants were associated with a single cancer phenotype, and five variants were associated with more than one cancer phenotype. Three variants were associated with age at onset of cancer and cancer status, one variant was associated with age at onset of cancer and age at onset of sarcoma, and one variant was associated with age at onset of sarcoma and cancer status. 71

103 72 Table 3.6: Summary of SOLAR association results for candidate gene variants Chr:Pos Gene p-value Beta SE Exonic function SIFT PolyPhen-2 RegulomeDB GERP Ref Alt MAF 1000G MAF Age at onset of cancer 16: PDIA NS D D C G : ATM NS D D C G : MEN Int.. 2b G C : MAP4K Int.. 1f 1.94 A G : ERCC2 < Int.. 2b 2.51 C T : ETV Int.. 2b 2.49 G A : TOP3A Int.. 1f G A : SMCR S.. 2b 5.20 G T : SHMT UTR3.. 1f G A : SHMT UTR3.. 1f 1.74 G C : SHMT Int.. 1f T C : MYBPC S.. 1f G A : MAP4K Int.. 1f 1.69 C T : LYPD S.. 1f A G Age at onset of sarcoma 8: RECQL S.. 2b 0.96 T C : MYBPC S.. 1f G A

104 Chr:Pos Gene p-value Beta SE Exonic function SIFT PolyPhen-2 RegulomeDB GERP Ref Alt MAF 1000G MAF Cancer status 16: PDIA NS D D C G : MEN Int.. 2b G C : RGS Int.. 2b 3.34 C T : ETV Int.. 2b 2.49 G A : HNF4A Int.. 2b 2.12 G A : GPT Int.. 2b G A : RECQL Int.. 2b G A : RECQL S.. 2b G A : MYBPC S.. 2b 0.53 G A : MRPL Int.. 2b G C : CDH Int.. 2b 3.50 T C : RECQL S.. 2b 0.96 T C Chr:Pos: Chromosome:Position. SE: Standard Error. SIFT: Sorting Intolerant from Tolerant score. PolyPhen-2: Polymorphism Phenotyping-2. GERP: Genomic Evolutionary Rate Profiling score. Ref: reference allele. Alt: alternate allele. MAF 1000G: Minor Allele Frequency in 1000 Genomes Project. MAF: Minor Allele Frequency in the study population. NS: nonsynonymous. S: synonymous. Int: intronic. UTR3: 3 untranslated region. UTR5: 5 untranslated region. D: deleterious..: not annotated in database. 73

105 Segregation analysis results Of the 23 different candidate variants identified, five variants were only seen in one family (PDIA2, ERCC2, HNF4A, MYBPC3 and MRPL28 ). However, using all three criteria for familial segregation, only one variant in the PDIA2 gene showed nominal association with age at onset of cancer and cancer status and complete familial segregation in family 2 (Figure 3.4). Each family member with cancer was heterozygous at this position, whereas unaffected family members were homozygous for the reference allele at this position. None of the other prioritised candidate gene variants showed complete familial segregation in any of the families according to the familial segregation criteria. 2-I-1 2-I-2 Prostate Key Affected male Affected female 2-III-1 2-II-1 2-II-2 2-II-3 Sarcoma Melanoma Melanoma 2-II-4 Unaffected male Unaffected female Proband Patient Genotype at position in PDIA2 gene Read depth Ref, alt Patient 2-I-1 C/C 206,1 Patient 2-I-2 C/G 109,135 Patient 2-II-1 C/G 74,67 Patient 2-II-2 C/G 63,43 Patient 2-II-3 C/G 106,108 Patient 2-II-4 C/C 138,0 Patient 2-III-1 C/C 161,0 Figure 3.4: Genotypes for the PDIA2 variant that shows segregation in patients with cancer in family 2 74

106 3.4.5 Evidence further supporting germline risk genes The nominally significant (p-value < 0.05) variants that showed familial segregation were researched using several in silico resources. Table 3.7 contains a combined summary of several in silico resources for all nominally significant candidate germline risk variants identified by the three prioritisation strategies that showed familial segregation and the genes in which they arise. Evidence from the table indicates that none of the candidate risk variants identified were reported in COSMIC (database of genes somatically mutated in cancers). However, six of the genes in which germline risk variants were identified were each reported to have mutations in the COSMIC database. None of the genes were listed in the COSMIC cancer gene census. They also were not listed in the PubMeth database, which suggests there is currently no evidence of methylation of these genes in cancer. Two of the genes were reported to have gene functions that support involvement in cancer pathogenesis in NCBI. The Gene References into Functions (GeneRIF) for ABCB5 suggests a role for this gene in chemoresistance and the GeneRIF for KIF2C suggests this gene is involved in directional migration and invasion of tumour cells. A summary of the PubMed searches for the eight candidate risk genes is summarised in Table 3.8. The PubMed searches revealed previously published associations between the ABCB5, KIF2C, and PDIA2 genes and cancer. However, there is no supporting evidence for the involvement of ARHGAP39, C16orf96, ZFP69B, UVSSA and BEAN1 genes in cancer pathogenesis at this time. The single publication returned by the search strategy for the ARHGAP39 gene revealed a role for the gene as a binding partner for CNK2 which is a spatial modulator of Rac cycling during spine morphogenesis. 297 This publication did not report any association of ARHGAP39 and cancer. The PubMed search for the BEAN1 gene returned results on randomised soya trials, labelled BEAN1 and BEAN2. 298, 299 No publications were returned on the function of the BEAN1 gene or involvement in cancer pathogenesis. 75

107 Table 3.7: Summary of findings from in silico resources investigating the role of candidate germline risk variants in cancer pathogenesis Gene Genomic Variant in No. Cancer SuperPath GO Molecular function Methylation GeneRIF location COSMIC mutations gene in COSMIC census ARHGAP39 8: No 246 No Developmental biology GTPase activator activity No No Signalling by Robo receptor 75 NTR receptor-mediated signalling Signalling by GPCR Signalling by Rho GTPases C16orf96 16: No 155 No.. No No ABCB5 7: No 332 No ABC-family proteins mediated ATP binding No Chemoresistance transport Xeonobiotic-transporting Transmembrane transport of ATPase activity small molecules Efflux transmembrane transporter activity ATPase activity

108 Gene Genomic Variant in No. Cancer SuperPath GO Molecular function Methylation GeneRIF location COSMIC mutations gene in COSMIC census ZFP69B 1: No 0 No Gene expression DNA binding Transcription factor activity, sequence-specific DNA binding Protein binding Metal ion binding No No UVSSA 4: No 0 No Transcription-coupled RNA polymerase II core No No nucleotide excision repair binding DNA double strand break Protein binding repair BEAN1 16: No 31 No.. No No KIF2C 1: No 141 No Golgi-to-ER retrograde Microtubule motor activity No Directional transport Cell cycle Mitotic metaphase and anaphase Mitotic prometaphase Protein binding ATP binding Microtubule binding ATPase activity migration and invasion of tumour cells Vesicle-mediated transport

109 Gene Genomic Variant in No. Cancer SuperPath GO Molecular function Methylation GeneRIF location COSMIC mutations gene in COSMIC census PDIA2 16: No 114 No Statin pathway Protein disulfide isomerase activity Steroid binding Protein binding Lipid binding Disulfide oxidoreductase activity No No Genomic location: chromosome:position. COSMIC: Catalogue of Somatic Mutations in Cancer database ( 134 No. mutations in COSMIC: the number of mutations reported in the gene in the COSMIC database. Cancer gene census: is the gene reported in the cancer gene census in COSMIC? The cancer gene census is a catalogue of genes for which mutations have been causally implicated in cancer. SuperPath: from Pathcards, an integrated database of human pathways and their annotations. ( 293 Human pathways are clustered into SuperPaths based on gene content similarity. GO molecular function: Gene Ontology molecular function. 294 Methylation: is the gene reported to be methylated in cancer by PubMeth? ( 295 GeneRIF: Gene References Into Functions from National Center for Biotechnology Information ( 296 Are any GeneRIF associated with cancer reported for the gene? Robo: Roundabout family of proteins. NTR: Neurotrophins. GPCR: G-protein-coupled receptors. GTPase: Guanosinetriphosphatase. ABC: ATP-binding cassette. ATP: Adenosine triphosphate. ATPase: Adenosinetriphosphatase. ER: endoplasmic reticulum.

110 Table 3.8: Summary of search results from PubMed for genes in which germline variants were identified Gene No. of publications Role of gene Selected references ARHGAP C16orf ABCB5 109 ABCB5 is a drug efflux pump associated with melanoma, colon cancer, Merkel cell carcinoma, oral squamous cell carcinoma, acute leukemia, colorectal cancer, hepatic cancer, breast cancer and osteosarcoma drug resistance. ABCB5 has also been found to be overexpressed at the transcriptional level in a number of cancer subtypes, including breast cancer, melanoma. Alterations found in ABCB5 reported in lung cancer. ZFP69B UVSSA 5 UVSSA is involved in transcription-coupled nucleotide excision repair by relieving RNA polymerase II arrest at damaged sites to permit repair of the template strand. Mutations in UVSSA associated with Cockayne syndrome group B (characterised by photosensitivity, growth failure, progressive neurodevelopmental disorder, and premature ageing but no predisposition to skin cancer) BEAN1 2.. KIF2C 55 KIF2C (also known as MCAK) is critical in the regulation of microtubule dynamics during mitosis. KIF2C is also involved in the directional migration and invasion of tumour cells and plays a role in cell proliferation. KIF2C is a gene likely to be involved in carcinogenesis

111 Gene No. of publications Role of gene Selected references PDIA2 3 Gene expression of PDIA2 found to influence the prognostic significance of TWIST (correlated with cancer invasion and metastasis in several human cancers). PDIA2 plays a role in the maintenance of endoplasmic reticulum homeostasis and endoplasmic reticulum stress-induced apoptosis. 324, 325 PubMed search was performed using a string ( gene name ) AND (cancer OR malignancy OR tumor* OR tumour* OR sarcoma) in April Abstracts were screened for relevance to the current study.

112 3.5 Discussion The filtering and prioritisation of eight germline variants generated by WES in three families were described in this chapter. Eight candidate germline risk variants were found to show nominal association with cancer and age at onset of cancer in two of the three cancer cluster families Variant filtering and prioritisation strategies The annotation results using ANNOVAR are consistent with a previous publication that reports a significant amount of DNA fragments across WES capture fall outside target regions. 256 There were slightly more synonymous variants than nonsynonymous variants, which is also consistent with previous findings. 286 SIFT and PolyPhen-2 scores from ANNOVAR annotation were used to determine if the variants were likely to have a deleterious effect on protein function. A previous study reports reasonable sensitivity for SIFT and PolyPhen-2 (69% and 68%, respectively) but low specificity (13% and 16%, respectively). 326 Therefore, both programs have a high false-positive rate and these results should be interpreted with caution and should be reported in the context of other available evidence. 326 In addition to variants reported as deleterious or tolerated by SIFT and PolyPhen-2, there are a number of variants that were not annotated with a score (unknown). In particular, 80% of variants prioritised by the rare variants strategy were filtered out because they were unknown in both databases. Although two of the prioritisation strategies identified more regulatory variants than structural variants, of the eight candidate risk variants that showed familial segregation, seven were structural, and only one was regulatory. In this study, exome sequencing combined with variant filtering and prioritisation is an efficient strategy for identifying risk alleles in cancer cluster families. 81

113 3.5.2 Association and segregation analyses of candidate risk variants in families Family segregation studies are re-emerging as an optimal way to classify extremely rare variants. 327 In this study, three assumptions were made in determining familial segregation. These assumptions did not take into account the possibility of unaffected carriers (incomplete penetrance), later onset of disease, or risk variants that occur in cases in more than one family. Therefore, some true variants may have been excluded using these assumptions. SOLAR was used to test for association of filtered and prioritised variants with both age at onset of disease and disease status. Despite efforts to filter and prioritise variants, no variants reached statistical significance after correcting for multiple testing and a nominal p-value of < 0.05 was therefore used to select variants for familial segregation analyses. The large number of variants identified and the relatively small sample size are the likely reasons that no variants reached statistical significance after correcting for multiple testing in this study. Despite these limitations, by treating each family as a separate discovery unit, it was hoped that some insight might be gained into genetic contributions to the risk of cancer in each family. Eight variants nominally associated with age at onset of cancer and cancer status were identified in two of the three cancer cluster families. The candidate risk variants identified in this study were all private variants, seen only in one family. There has been increasing awareness that rare variants of modest to large effect contribute to complex diseases and may explain a substantial proportion of missing heritability. 129 There has been, therefore, a return to family-based studies to identify rare risk variants involved in common human 133, disease. Recent sequencing studies have shown that the rate of private mutations in individuals is larger than previously expected Rare, private mutations found in families could be due to the explosion of human populations and the slowing of negative selection by improved food supplies, sanitation, vaccines 82

114 and routine health care Rare variants that are private to families could constitute a proportion of disease risk variants. 328 It is plausible that the variants found in the ABCB5, KIF2C and PDIA2 genes may be involved in the pathogenesis of cancer based on previous publications and the proposed function of the protein. Each of these genes is discussed in more detail below The ABCB5 gene The ABCB5 gene is a ATP-binding cassette (ABC) drug efflux transporter present in a number of stem cells. 332, 333 ABCB5 functions as a determinant of membrane potential and regulator of cell fusion in physiologic skin cells. 334 This gene is also expressed in clinical malignant melanoma tumours and preferentially marks CD133 + stem cell phenotype expressing tumour cells. 334 ABCB5 is a rhodamine-123 efflux transporter and marks CD133-expressing progenitor cells. ABCB5 regulates membrane potential in these progenitor cells and determines the propensity to undergo cell fusion. 334 Membrane hyperpolarisation is associated with the multidrug resistance phenotype of human cancer cells. 335 ABCB5 plays a role in multi-drug resistance of multiple malignancies including human malignant melanoma, 333, 336, 337 colon cancer, 304, 338 Merkel cell carcinoma, 305 oral squamous cell carcinoma, 306 acute leukaemia, 307 colorectal cancer, 309 hepatocellular carcinoma, 310 breast cancer, 311 and osteosarcoma. 303 Melanoma is resistant to the effects of doxorubicin, 333 a chemotherapy drug used to treat many different types of cancer. It has been proposed that the ABCB5 drug efflux function may be involved in doxorubicin resistance. 334 The variant identified in the ABCB5 gene may be phenotypically relevant to family 2 as this family has two family members affected by melanoma (Patient 2-II-2 and Patient 2-II-3), in addition to a prostate cancer case (Patient 2-I-1), and a sarcoma case (Patient 2-II-1). 83

115 The KIF2C gene KIF2C is a kinesin-like protein that functions as a microtubule-dependent molecular motor. 339 The KIF2C gene (also known as MCAK), is one of the best characterised members of the kinesin-13 family and plays an important role in microtubule dynamics during mitosis. 320 The deregulation of KIF2C induces defects in spindle assembly, chromosome congression and segregation leading to chromosome instability, one of the hallmarks of cancer. 320 KIF2C is important for the migration and invasion of tumour cells via the modulation of microtubule dynamics in the 320, 345, 346 cytoskeleton. The KIF2C gene has been identified as a tumour antigen in patients with colorectal cancer. 347 The overexpression of KIF2C associates with a more invasive and metastatic phenotype and poor prognosis for breast, gastric and colorectal cancer patients KIF2C may represent an attractive target for antigen-specific 347, 348 immunotherapies in colorectal cancer and other malignancies The PDIA2 gene The PDIA2 gene is the pancreas-specific member of the protein disulphide isomerase (PDI) family of proteins. PDIA2, as with other PDIs, has a central role as a reductase, an oxidase, an isomerase and molecular chaperone in the endoplasmic reticulum. 351 It has been proposed that PDIA2 plays a role in the production and secretion of digestive enzymes in vivo 352 and in the binding and regulation of oestrogen synthesis. 353 A higher level of PDIA2 expression was found to be associated with shorter survival time in patients whose prostate cancer expressed a high level of TWIST but not in patients whose prostate cancer expressed a low level of TWIST. 324 TWIST is an oncogene that is correlated with cancer invasion and metastasis in human cancers including breast cancer, rhabdomyosarcoma, gastric carcinomas, bladder and prostate cancer Little is known about the role of PDIA2 in prostate cancer, although lower levels of PDIA2 expression were associated with better survival. 324 Therefore, PDIA2 may promote cancer progression. 324 However, PDIA2 alone was a poor prognostic marker for prostate cancer

116 3.5.3 Conclusion In conclusion, WES data was annotated, filtered and prioritised in an attempt to identify candidate germline risk variants that may be involved in cancer or sarcoma pathogenesis in three cancer cluster families. As there is no gold standard for the filtering and prioritisation of WES data, these results represent the current state of tools, databases and knowledge of cancer biology. With the data obtained, it is not possible to determine whether the variants in the ARHGAP39, C16orf96, ZFP69B, UVSSA and BEAN1 genes are pathogenic mutations. These genes, however, become candidates that can be further tested for association with cancer in independent families and study populations. With further genetic evidence of involvement in risk of cancer, functional studies including assays of patient-derived tissue or well-established cell or animal models of gene function could be undertaken to determine the causal effect of all candidate risk variants on the cancer phenotype. 236 Due to time and budget limitations, these types of functional studies are beyond the scope of this thesis. 85

117 86

118 Chapter 4 Aim 3: A comparison of matched tumour and germline DNA from two sarcoma patients 4.1 Introduction Next Generation Sequencing (NGS) of tumour samples and matched germline samples is a powerful strategy for studying the genetic basis of cancer initiation, development, and growth. 133 The third aim of this study was to perform a matched tumour and germline analysis on two myxoid liposarcoma patients using peripheral blood genomic DNA and genomic DNA isolated from tumour tissue to identify somatic mutations Myxoid liposarcoma Myxoid liposarcomas are the second most common group of adipocytic/lipogenic sarcomas. 64 Myxoid liposarcomas are malignant tumours composed of uniform round to oval shaped primitive non-lipogenic cells and a variable number of small signet-ring cell lipoblasts. 64 The tumours typically exhibit a FUS-DDIT3 or EWSR1-DDIT3 rearrangement. 64 Myxoid liposarcomas occur most commonly in the deep soft tissue of the extremities and very rarely in the retroperitoneum. 87

119 Somatic variants A comparison of matched tumour and germline samples from a patient allows researchers to distinguish between somatic variation (< 0.01% of variants) and inherited germline variation (> 99.99% of variants). 133 Germline variants are those that exist in the germline DNA which is the source of DNA for all cells in the body. 149 A variant contained within the germline can be passed from parent to offspring. The identification of putative germline variants was the focus of Aim 2 (Chapter 3). Therefore, germline variants will not be reported in this chapter. In contrast, somatic variants are those found in the tumour DNA but not in the germline DNA. 149 Most cancers arise and evolve as a consequence of somatic mutations. 358 The characterisation of somatic mutations in cancer genomes is essential for understanding the disease and for the development of targeted therapeutics. 359 Over the last three decades, more than 600 genes have been 134, 358 shown to be somatically mutated in cancers. Molecular characterisation of somatic driver mutations allows greater understanding of biological abnormalities within cancer cells and provides information on the function of gene products, and relationships between genes and biochemical pathways. 134 Development of new therapeutic and preventative agents are dependent on the identification and modulation of these molecular targets. 134, 360 Targeted therapies for advanced lung cancer, 361 melanoma, 362 colorectal cancer, 363 and gastrointestinal stromal tumour 364 are examples that have resulted from the translation of knowledge gained from genomics. In addition to somatic variants, a comparison of matched tumour and germline DNA can also identify the absence of heterozygosity at loci in tumour DNA compared to germline DNA Loss of heterozygosity Loss of heterozygosity (LOH) is a common genetic event in cancer development. 365 LOH is a change in the polymorphic markers from a heterozygous state in the germline DNA to a homozygous state in the tumour DNA. 366 In cancers, the absence of one functional copy of a tumour suppressor gene does not affect the phenotype. However, if LOH occurs and the remaining normal copy of the tumour 88

120 suppressor gene is lost, this will result in the complete loss of the protective function of the tumour suppressor gene. LOH is known to be involved in the somatic loss of wild-type alleles in many inherited cancer syndromes such as 366, 367 retinoblastoma and hereditary breast and ovarian cancer syndromes Somatic copy number alteration In addition to distinguishing somatic and LOH variants, somatic copy number alterations (SCNA) can be identified in a tumour sample relative to the matched germline sample by comparing the normalised read depth. 368, 369 The DNA sequence copy number is the number of copies of DNA in a region of a genome. 370 Cancer progression often involves alterations in DNA copy number. 370 In humans, the normal copy number is two for all the autosomes. A copy number variation (CNV) is defined as structurally variant regions where copy number differences have been observed between two or more genomes that are larger than one kilobase (kb) in size. 371 CNVs can alter transcription of genes by changing the dosage or by disrupting proximal or distant regulatory regions. 372 SCNA, distinguished from germline CNV, play a role in activating oncogenes and inactivating tumour suppressor genes. 13 Identification of SCNA can provide valuable insights into the cellular defects that cause cancer and suggest potential therapeutic strategies. 373 SCNA and CNVs have a significant role in tumourigenesis in many cancers including gastric cancer, 374 ovarian cancer, 375 hepatocellular carcinoma, 376 testicular germ cell tumours, 377 colorectal carcinoma, 378 and bladder cancer. 379 The characterisation of focal SCNAs has led to the identification of novel cancer genes such as MYB, PAX5 and DUSP Bioinformatic assessment of matched tumour and germline samples A number of bioinformatic tools have been developed to analyse matched tumour and germline samples. Initially, these tools used algorithms that involved calling variants in the tumour and germline samples separately followed by classification using a statistical significance test or simple subtraction. 388 More recently, tools have been developed that compare the tumour and germline directly at each 89

121 locus. VarScan2 and Strelka are two calling algorithms that were specifically 368, 369, 389 designed for the joint analysis of matched tumour and germline samples. VarScan2 uses tumour and germline samples to heuristically detect sequence 368, 369 variants and classify them by somatic status (germline, somatic or LOH). Strelka utilises a novel Bayesian approach to represent continuous allele frequencies for both tumour and normal samples to efficiently identify somatic variants. 389 Using Strelka, the normal sample is represented as a mixture of diploid germline variation with noise, and the tumour sample is represented as a combination of the normal sample with somatic variation. 389 It is important to identify somatic mutations in cancer studies as these variants often play important roles in tumour development and treatment decisions Somatic mutations and drug sensitivity The identification of somatic driver mutations that arise in tumours is important in developing new cancer therapeutic targets as genetic variation influences the response of an individual to drug treatments. 390 The current treatment for most cancers includes using cytotoxic chemotherapy, which is not precisely targeted to the somatic mutations that drive malignant transformation. 390 Somatic mutations can influence tumour behaviour and clinical outcome. Therefore, therapies should be targeted to the patient s tumour genotype rather than a generic treatment. An increased understanding of somatic mutations in individual patients has the potential to make therapies safer and more effective by assisting treatment selection and dosage based on driver mutations in the tumour. The Genomics of Drug Sensitivity in Cancer database ( org/) 391 is a large dataset on drug sensitivity in cancer cells linked to genomic information to facilitate the discovery of new biomarkers of drug response. 391 The database contains information on over 250 anticancer drugs across > 1,000 cell lines. 391 Molecular markers are identified by integrating data from the Catalogue of Somatic Mutations in Cancer (COSMIC) database 134 and cell line drug sensitivity data. 90

122 4.1.4 Outline of chapter Whole exome sequencing (WES) was performed on matched tumour and germline DNA from two myxoid liposarcoma patients from the families described in Chapter 2. VarScan2 was used to identify candidate somatic variants that were confirmed using Strelka. VarScan2 was also used to identify LOH variants and SCNA events to determine regions of interest in both patients. 4.2 Methods Whole exome sequencing Tumour DNA from formalin-fixed and paraffin-embedded (FFPE) tumour samples and germline DNA from Patient 1-II-2 and Patient 2-II-1 were available to perform a matched tumour-germline analysis. DNA was extracted at the Peter MacCallum Cancer Centre in Melbourne, Australia. After microdissection of tumour material from FFPE tissue, DNA was extracted using a DNeasy Tissue kit (Qiagen) as previously described. 392 Anti-coagulated blood was processed using a Ficoll gradient. DNA was extracted from the nucleated cell product using QIAamp DNA blood kit (Qiagen). Patient 1-II-2 (Figure 4.1) is a male patient who was diagnosed with a myxoid liposarcoma at 39 years of age. Patient 2-II-1 (Figure 4.2) is a male patient who was diagnosed with a myxoid liposarcoma at 61 years old. 91

123 1-I-1 1-I-2 Key Affected male Affected female 1-II-1 * 1-II-2 Sarcoma 1-II-3 Unaffected male Unaffected female Proband 1-III-1 Sarcoma 1-III-2 * Patient selected for tumour-normal analysis Figure 4.1: Pedigree of family 1 highlighting sarcoma Patient 1-II-2 for tumour-germline comparison 2-I-1 2-I-2 Prostate Key Affected male Affected female Unaffected male * 2-III-1 2-II-1 Sarcoma 2-II-2 Melanoma 2-II-3 Melanoma 2-II-4 Unaffected female Proband * Patient selected for tumour-normal analysis Figure 4.2: Pedigree of family 2 highlighting sarcoma Patient 2-II-1 for tumour-germline comparison 92

124 Due to difficulties performing WES on older FFPE samples, 393 DNA extracted from these samples were sent to an external sequencing facility. The four samples were sequenced using Agilent SureSelect V5 Capture on the Illumina HiSeq 4000 at 60X coverage Pre-processing and quality control FASTQ files were received from Macrogen, Inc. Initial quality control (QC) reports were generated using FastQC (version ), a quality control application for high throughput sequence data. 394 FastQC reads FASTQ files and can either provide an interactive application to review the results of several different checks or create an HTML based report which can be integrated into a pipeline. 394 QC reports were generated on sequence quality, GC content, duplication levels and adapter content Adapter trimming The presence of technical sequences such as adapters in WES data can result in suboptimal downstream analyses. 395 The Illumina-specific adapter sequences were trimmed from the FASTQ files using Trimmomatic (version 0.36). 395 As Illumina sequences are paired-end, the palindrome mode was used. This mode is specifically aimed at detecting typical adapter read-through situations in which the DNA fragment is shorter than the read length and indicates adapter contamination on the end of the reads. 395 After the Illumina-specific adapters had been trimmed from the FASTQ files, the second round of QC reports were generated on the adapter trimmed data using FastQC Sequence alignment and calling The raw sequencing data was then aligned to the human genome using the Burrows-Wheeler Aligner (BWA, version 0.7.2). 396 BWA alignment was performed in two steps. In the first step, the genome was indexed to the human genome build 19 (hg19) reference sequence. In the second phase, BWA Maximal Exact Matches (BWA-MEM) was used to run the alignment to align the sequence reads to hg19. 93

125 The alignment step creates the alignment in Sequence Alignment/Map (*.sam) format. SAMtools (version 1.3.1) View 397 was used to convert the *.sam files to *.bam format to reduce the size of the data. Summary statistics were created for the *.bam files using SAMtools flagstat. 397 Index files were created for each *.bam file using SAMtools index. 397 Local realignment was performed on the *.bam files in two stages using Genome Analysis Toolkit (GATK) RealignerTargetCreator and IndelRealigner (version 3.4.0). 180 The Picard (version 2.4.1) FixMateInformation tool was used to ensure that all read entries had their mate information written correctly. The Picard MarkDuplicates tool was then used to identify duplicate reads BAM quality control A final round of QC was performed on the *.bam files using GATK DepthOfCoverage, 180 and Picard CollectInsertSizeMetrics and CollectAlignmentSummaryMetrics to determine coverage, insert size (the library portion between the adapter sequences) and alignment metrics, respectively Generate mpileup file The germline and tumour *.bam files for each patient were grouped using SAMtools mpileup. 181 Alignment records were consolidated by sample identifiers in read group header lines Somatic variant calling using VarScan2 The genotype for each sample was determined from mpileup files using VarScan2 (version 2.3.9). 368 The algorithm read the data from both tumour and germline samples simultaneously. VarScan2 employed a heuristic approach to call variants that met the thresholds for read depth, base quality, variant allele frequency, and statistical significance. 368, 369 If the genotypes did not match, the read counts were evaluated by one-tailed Fisher s exact test in a two-by-two table, comparing the number of reference-supporting reads and variant-supporting reads observed in the tumour to the numbers that were observed in the germline. 368 If the 94

126 resulting p-value met the significance threshold (default 0.10), then the variant was called somatic (if the germline matched the reference genome at that position). 368 The VarScan2 subcommand, processsomatic, was then used to create output files of somatic variants based on confidence (low confidence and high confidence). High confidence variants are classed as those with a tumour variant allele frequency > 15%, normal variant allele frequency < 5%, and a somatic p-value of < The remaining variants are classed as low confidence. VarScan2 somaticfilter was used to filter the possible false positives from the high confidence somatic mutations. Table 4.1 shows the settings used to run the somaticfilter command. Table 4.1: Parameters specified for VarScan2 somaticfilter to filter false positives from the high confidence somatic mutations Parameter Specified Minimum read depth 10 Minimum supporting reads for a variant 2 Minimum number of strands on which variant observed 1 Minimum average base quality for variant-supporting reads 20 Minimum variant allele frequency threshold 0.2 Default p-value threshold for calling variants 1 x 10 1 Bonferroni adjustments were made to the somatic p-value values from VarScan2 to correct for multiple testing. 292 The total number of variants in the mpileup files for each patient were used for the correction. The genotypes of any significant variants were visually confirmed by importing *.bam files into Integrative Genomics 183, 184 Viewer (IGV, version ) by determining the number of reads for each allele Somatic variant calling using Strelka A second somatic variant caller, Strelka (version ), 389 was used to confirm the statistically significant somatic variants called by VarScan2. The first step of somatic variant analysis using Strelka is to run preliminary configuration validation (ensure that the chromosome names match in the *.bam header and 95

127 reference genome). Template configuration files from Strelka were used in this analysis. The configuration generates a makefile that controls the analysis step. The second phase is to run the analysis using the makefile. The sorted tumour and germline *.bam files and hg19 reference sequence were used in the analysis Evidence further supporting somatic risk genes The significant somatic variants and the genes in which they arise were further examined for evidence in cancer pathogenesis using several in silico resources including COSMIC (catalogue of somatic mutations), 134 the pathway unification database (PathCards), 293 gene ontology (GO) annotations, 294 PubMeth (a database of methylation in cancer), 295 and National Center for Biotechnology Information (NCBI). 296 A PubMed search was performed using a string ( gene name ) AND (cancer OR malignancy OR tumor* OR tumour* OR sarcoma) in April Abstracts were screened for relevance to the current study Drug sensitivity The genes in which somatic mutations were identified in two sarcoma patients were searched in the Genomics of Drug Sensitivity in Cancer database ( 391 to determine whether they were known molecular targets Loss of heterozygosity variant calling using VarScan2 VarScan2 was used to call LOH variants. Similar to the somatic variant calling process, if the genotype between tumour and germline DNA did not match, the read counts were evaluated by a one-tailed Fisher s exact test. If the resulting p-value met the significance threshold (default 0.10), then the variant was called LOH (if the germline was heterozygous). Bonferroni adjustments were made to the LOH p-value values from VarScan2 to correct for multiple testing. 292 The total number of variants in the mpileup files for each patient were used for the correction. The genotypes of any significant variants were visually confirmed by importing *.bam files into IGV. 96

128 Variant annotation and filtering Statistically significant somatic and LOH variants were annotated using Annotate Variation (ANNOVAR, version 2015Jun16) 245 and Regulome database (RegulomeDB). 257 The somatic and LOH variants that reached statistical significance after Bonferroni correction were cross-referenced to the exclusion list of Fuentes Fajardo et al. (2012) (Available in the paper s Supplementary material: Table S7 gene exclusion list final ) to determine if any variants in highly polymorphic regions should be excluded Somatic copy number analysis using VarScan2 VarScan2 copynumber was applied to the tumour-germline mpileup files to create a single output file of raw SCNAs. VarScan2 copycaller was then used to adjust for GC content and make preliminary calls. The adjusted calls files were imported into R, 281 and the package DNAcopy (version ) 398 was used to perform circular binary segmentation on a per-chromosome basis to smooth and segment the raw output from VarScan2 copycaller. 370 The results of DNAcopy were plotted in R to visualise SCNA. 4.3 Results Whole exome sequencing Raw data reports from Macrogen, Inc. are summarised in Table 4.2. The GC content for an exome typically falls within the range of 49-51%. 399 Therefore, the samples show just below average %GC content, with the tumour samples showing lower %GC content than the germline samples. Three of the samples have over 90% bases with a base quality (Q) score above 20 in the Phred scale (call accuracy of 99%), except Patient 2-II-1 tumour sample, which has 88.6% bases with a Q score above 20. Pre-processing QC and adapter trimming did not result in any sequences being flagged or trimmed. 97

129 Table 4.2: Raw data summary from Macrogen Inc. for Patient 1-II-2 and Patient 2-II-1 germline and tumour samples Sample ID Total read bases (base pairs) Total reads GC(%) AT(%) Q20(%) Q30(%) Patient 1-II-2 germline 7,720,761,786 76,443, Patient 2-II-1 germline 6,079,509,766 60,193, Patient 1-II-2 tumour 6,711,209,620 66,447, Patient 2-II-1 tumour 5,022,136,524 49,724, Sample ID: sample name. Total read bases: total number of bases sequenced. Total reads: total number of reads. GC(%): GC content. AT(%): AT content. Q20(%): Ratio of reads that have Phred quality score of over 20. Q30(%): Ratio of reads that have Phred quality score of over Sequence alignment and calling Summary statistics on the trimmed *.bam files were computed using Samtools flagstat 181 and are presented in Table 4.3. The results show that both germline samples had over 99% of reads mapped, and both tumour samples had over 98% of reads mapped. Both germline samples had almost all of the mapped reads properly paired (> 98.8%). However, the tumour samples had slightly lower properly paired reads (93.68% for Patient 1-II-2 and 95.86% for Patient 2-II-1). 98

130 Table 4.3: Summary statistics generated using Samtools flagstat for Patient 1-II-2 and 2-II-1 germline and tumour samples Statistic Patient 1-II-2 germline Patient 2-II-1 germline Patient 1-II-1 tumour Patient 2-II-1 tumour Total (QC-passed reads + QC-failed reads) 76,335,490 60,113,342 63,327,174 49,372,458 Duplicates Mapped (%) 75,948,149 (99.49%) 59,803,630 (99.48%) 62,074,975 (98.02%) 48,478,673 (98.19%) Paired in sequencing 76,335,490 60,113,342 63,327,174 49,372,458 Read 1 38,167,745 30,056,671 31,663,587 24,686,229 Read 2 38,167,745 30,056,671 31,663,587 24,686,229 Properly paired 75,457,268 (98.85%) 59,445,380 (98.89%) 59,321,774 (93.68%) 47,327,518 (95.86%) With itself and mate mapped 75,833,239 59,704,772 61,514,992 47,943,636 Singletons (%) 114,910 (0.15%) 98,858 (0.16%) 559,983 (0.88%) 535,037 (1.08%) Mate mapped to a different chromosome 174, , ,018 39,240 Mate mapped to a different chromosome (mapq 5) 156,396 93,541 65,912 27,128 QC: quality control.

131 Local realignment was performed using GATK RealignerTargetCreator. For Patient 1-II-2 there were 3,793,051 (2.72%) reads filtered out during the traversal. Of these, 224,019 reads failed the bad mate filter, 3,569,018 reads failed the mapping quality zero filter, and 14 reads failed the unmapped read filter. For Patient 2-II-1 there were 3,060,049 (2.79%) reads filtered out during the traversal. Of these, 121,551 reads failed the bad mate filter, 2,938,469 reads failed the mapping quality zero filter, and 29 reads failed the unmapped read filter. For Patient 1-II-2, no reads were filtered out of 76,335,490 total reads in the germline sample, and no reads were filtered out of 63,327,174 total reads in the tumour sample. For Patient 2-II-1, no reads were filtered out of 60,113,342 total reads in the germline sample, and no reads were filtered out of 49,372,458 total reads in the tumour sample BAM quality control GATK depth of coverage results are presented in Figure 4.3. As expected, the majority of bases were covered at a depth of 100X or less in each sample. Germline samples (blue) for both patients show slightly higher coverage compared to the tumour samples (orange). 100

132 220, , , , , ,000 Number of bases 145, ,000 95,000 Number of bases 145, ,000 95,000 70,000 70,000 45,000 45,000 20,000 20,000-5,000 >=0 >=50 >=100 >=150 >=200 >=250 >=300 >=350 >=400 >=450 >=500 Depth Patient 1-II-2 Germline Patient 1-II-2 Tumour -5,000 >=0 >=50 >=100 >=150 >=200 >=250 >=300 >=350 >=400 >=450 >=500 Depth Patient 2-II-1 Germline Patient 2-II-1 Tumour (a) Patient 1-II-2 (b) Patient 2-II-1 Figure 4.3: Genome analysis toolkit depth of coverage summary for Patient 1-II-2 and Patient 2-II-1 germline and tumour DNA

133 The average insert size for the germline samples of both Patient 1-II-2 and Patient 2-II-1 is approximately 150 base pairs. The tumours samples have slightly smaller insert sizes of approximately 125 base pairs and 140 base pairs for Patient 1-II-2 and Patient 2-II-1, respectively. Figure 4.4 shows histogram plots of the insert size distribution for both patients germline and tumour samples generated by Picard (Patient 1-II-2: top panels, Patient 2-II-1: bottom panels). a) b) Patient 1-II-2 germline Patient 1-II-2 tumour c) d) Patient 2-II-1 germline Patient 2-II-1 tumour Figure 4.4: Insert size histogram plots generated by Picard for Patient 1-II-2 and Patient 2-II-1 germline and tumour samples 102

134 High level metrics about the alignment of reads within a *.bam file were produced by the CollectAlignmentSummaryMetrics tool from Picard. All the reads from both patients germline and tumour samples passed the filter criteria, and the percentage of reads aligned was above 98% for all samples Somatic variant calling VarScan2 VarScan2 identified 4,888 somatic variants in Patient 1-II-2, of which, 702 were classed as high confidence. Patient 2-II-1 had 2,667 somatic variants with 595 classed as high confidence. The results of the somaticfilter command (to remove possible false positives) for the SNV somatic high confidence files are presented in Table 4.4. Most of the variants that were removed from both patients failed the Reads2 requirement (minimum supporting reads for a variant). Table 4.4: Results from VarScan2 somaticfilter to remove possible false positives from the high confidence somatic calls for Patient 1-II-2 and Patient 2-II-1 Filter Patient 1-II-2 Patient 2-II-1 Total variants in input Coverage requirement (10) 3 7 Reads2 requirement (2) VarFreq requirement (0.2) 0 0 p-value requirement (1 x 10 1 ) 2 14 SNP clusters requirement 0 4 Near INDELs 0 0 Passed Reads2: minimum supporting reads for a variant filter. VarFreq: Minimum variant allele frequency filter. SNP: single nucleotide polymorphism. INDEL: insertion or deletion. 103

135 Bonferroni adjustment was performed on the p-values from VarScan2 to correct for multiple testing. As Patient 1-II-2 had 66,265,606 positions for comparison, the significance level after Bonferroni correction was α < 7.55 x After correcting for multiple testing, Patient 1-II-2 had 11 statistically significant somatic variants. Patient 2-II-1 had 67,054,165 positions for comparison, therefore the significance level after Bonferroni correction was α < 7.46 x After correcting for multiple testing, Patient 2-II-1 had three statistically significant somatic variants Validation of somatic variants using Strelka Of the 11 somatic variants identified by VarScan2 in Patient 1-II-2, ten were also reported as somatic variants by Strelka (Table 4.5). A variant in the CCDC66 gene (position 19: ) was reported by VarScan2 but not reported by Strelka. All three somatic variants identified by VarScan2 in Patient 2-II-1 were confirmed by Strelka (Table 4.6). The variants were annotated and cross-referenced against a provisional gene exclusion list, but no variants were removed

136 Table 4.5: Somatic variants identified by VarScan2 and Strelka for Patient 1-II-2 Chr:Pos Gene p-value Function SIFT PolyPhen-2 RegulomeDB GERP Ref Alt chr14: PRMT x NS D B G A 93,76 chr9: ASPN 5.55 x NS T D T A 60,51 chr6: LAMA x NS T D G A 67,49 chr4: TET x NS T B C T 61,55 chr18: FHOD x NS T D A T 54,54 chr19: GATAD2A 3.78 x NS T D G A 9,17 chr14: ADSSL x S.. 2b. C T 41,34 chr3: P4HTM 5.26 x NS. B 2b A C 54,26 chr11: SLC22A20,POLA x IG.. 5. G T 21,15 chr9: ABL x S.. 5. G C 53,32 Chr:pos: Chromosome:position. p-value: Fisher s p-value. SIFT: Sorting Intolerant From Tolerant. PolyPhen-2: Polymorphism Phenotyping-2. GERP: Genomic Evolutionary Rate Profiling score (a positive GERP score represents a substitution deficit, while a negative GERP score represents a substitution surplus). Ref: reference allele. Alt: alternate allele. NS: nonsynonymous. S: synonymous. IG: intergenic. D: deleterious. B: benign. T: tolerated. Read depth Ref, alt

137 Table 4.6: Somatic variants identified by VarScan2 and Strelka for Patient 2-II-1 Chr:Pos Gene p-value Function SIFT PolyPhen-2 RegulomeDB GERP Ref Alt chr8: SDR16C6P,PENK 9.39 x Intergenic.... C T 44,32 chr5: SLC6A x Splicing G A 10,12 chr5: PLK x Intronic.. 4. T C 31,23 Chr:pos: Chromosome:position. p-value: Fisher s p-value. SIFT: Sorting Intolerant From Tolerant. PolyPhen-2: Polymorphism Phenotyping-2. GERP: Genomic Evolutionary Rate Profiling score (a positive GERP score represents a substitution deficit, while a negative GERP score represents a substitution surplus). Ref: reference allele. Alt: alternate allele. D: deleterious. B: benign. Read depth Ref, alt

138 Evidence further supporting somatic risk genes Table 4.7 contains a summary of several in silico resources for the somatic risk variants and the genes in which they arise for both patients. None of the somatic risk variants were reported in COSMIC. However, all but one of the genes (SDR16C6P) were reported to have mutations in the COSMIC database. Two genes were listed in the COSMIC cancer gene census (TET2 and ABL1 ). The ABL1 and PENK genes were reported in the PubMeth database. However, none of the other genes were reported in PubMeth, which suggests there is currently no evidence of methylation of these genes in cancer. Evidence from NCBI suggests that ten genes have been reported to have gene functions that support involvement in cancer. Of these ten genes, six genes (PRMT5, LAMA2, TET2, FHOD3, ABL1 and PLK2 ) were reported to have Gene References into Functions (GeneRIF) evidence for involvement in cancer pathogenesis. Two genes (POLA2 and PENK) had GeneRIF that indicated these genes might be biomarkers for cancer and two genes (ASPN and P4HTM ) had GeneRIF that suggested these genes may be targets for cancer therapeutics. A summary of the PubMed searches for the candidate somatic risk genes is summarised in Table 4.8. The PubMed searches revealed previously published associations between the genes and cancer except for the ADSSL1 and SDR16C6P genes. Therefore there is no evidence supporting the involvement of ADSSL1 and SDR16C6P genes in cancer pathogenesis at this time. 107

139 Table 4.7: Summary of findings from in silico resources investigating the role of somatic risk variants and the genes in which they arise in cancer pathogenesis Gene Genomic Variant in No. Cancer SuperPath GO Molecular function Methylation GeneRIF location COSMIC mutations gene in COSMIC census PRMT5 chr14: No 95 No Regulation of TP53 Core promoter No Colorectal cancer activity sequence-specific DNA pathogenesis Chromatin organisation binding Acute myeloid Gene expression Transport of the SLBP independent mature mrna RNA transport Transcription corepressor activity Protein binding Methyltransferase activity Methyl-CpG binding leukemia growth Marker of poor prognosis in nasopharyngeal carcinoma Marker for early colorectal carcinomas ASPN chr9: No 96 No ECM proteoglycans Protein kinase inhibitor No Role in gastric cancer Degradation of the activity Therapeutic target extracellular matrix Calcium ion binding molecule Collagen binding

140 Gene Genomic Variant in No. variants Cancer SuperPath GO Molecular function Methylation GeneRIF location COSMIC in COSMIC gene census LAMA2 chr6: No 854 No Integrin pathway Receptor binding No Mutations in ERK signalling Arrhythmogenic Structural molecule activity hepatocellular carcinoma patients right ventricular cardiomyopathy Dilated cardiomyopathy Focal adhesion TET2 chr4: No 2,726 Yes Activated PKN1 Sulfonate dioxygenase No Involved in stimulates transcription activity leukemogenesis of androgen receptor regulated genes Chromatin regulation / Acetylation Gene expression DNA binding Protein binding Ferrous iron binding Zinc ion binding Oncogenic role in myeloid tumour FHOD3 chr18: No 393 No. Actin binding Protein binding No Glioma linear migration Associated with acute lymphoblastic leukemia Promotes invasive migration and local invasion in vivo

141 Gene Genomic Variant in No. variants Cancer SuperPath GO Molecular function Methylation GeneRIF location COSMIC in COSMIC gene census GATAD2A chr19: No 95 No Activated PKN1 stimulates transcription of androgen receptor regulated genes Chromatin organisation Gene expression Regulation of TP53 activity ADSSL1 chr14: no 114 No Purine metabolism Purine nucleotides de novo biosynthesis Metabolism purine metabolism Alanine, aspartate and glutamate metabolism Contributes to RNA polymerase II regulatory region sequence-specific DNA binding Transcription factor activity, sequence-specific DNA binding Protein binding Zinc ion binding Protein binding, bridging Magnesium ion binding GTPase activity Adenylosuccinate synthase activity GTP binding Ligase activity No. No. P4HTM chr3: No 75 No. Iron ion binding Calcium ion binding Oxidoreductase activity No May aid the design of novel therapies for inhibiting bone tumours

142 Gene Genomic Variant in No. variants Cancer SuperPath GO Molecular function Methylation GeneRIF location COSMIC in COSMIC gene census SLC22A20 chr11: No 83 No. Inorganic anion exchanger activity No. Sodium-independent organic anion transmembrane transporter activity POLA2 chr11: No 108 No Telomere C-strand DNA binding synthesis DNA-directed DNA E2F mediated regulation polymerase activity of DNA replication Protein heterodimerisation Regulation of activated activity PAK-2p34 by proteasome mediated degradation Purine metabolism Cell cycle, Mitotic No Prognostic biomarker in non small cell lung cancer pathogenesis

143 Gene Genomic Variant in No. variants Cancer SuperPath GO Molecular function Methylation GeneRIF location COSMIC in COSMIC gene census ABL1 chr9: No 1,684 Yes DNA double-strand breakmagnesium ion binding repair Development Slit-Robo signalling Regulation of actin dynamics for phagocytic cup formation DNA binding Actin monomer binding Nicotinate-nucleotide adenylyltransferase activity Protein kinase activity Cell cycle ErbB signalling pathway Yes BCR/ABL oncogene in leukaemia Promote breast cancer osteolytic metastasis Progression of gastric cancer SDR16C6P chr8: No. No.. No. PENK chr8: No 163 No Apoptotic pathways in Opioid peptide activity Yes Promoter methylation synovial fibroblasts GPCR pathway ERK signalling Nanog in Mammalian Neuropeptide hormone activity Opioid receptor binding associated with colorectal adenocarcinoma diagnosis ESC Pluripotency CREB Pathway

144 Gene Genomic Variant in No. variants Cancer SuperPath GO Molecular function Methylation GeneRIF location COSMIC in COSMIC gene census SLC6A18 chr5: No 186 No Transport of glucose and other sugars, bile salts and organic acids, metal ions and amine compounds Amino acid transport across the plasma membrane Neurotransmitter:sodium symporter activity Amino acid transmembrane transporter activity Symporter activity No. PLK2 chr5: No 153 No FoxO signalling pathway Nucleotide binding No Promoting tumour Gene expression Protein kinase activity progression TP53 Regulates transcription of cell cycle genes DNA damage Protein serine/threonine kinase activity Signal transducer activity Protein binding Increases cell proliferation and decreases apoptosis in gastric cancer cells Regulation of TP53 activity Genomic location: chromosome:position. COSMIC: Catalogue of Somatic Mutations in Cancer database. 134 No. mutations in COSMIC: the number of mutations reported in the gene in the COSMIC database. Cancer gene census: is the gene reported in the cancer gene census in COSMIC? The cancer gene census is a catalogue of genes for which mutations have been causally implicated in cancer. SuperPath: from Pathcards, an integrated database of human pathways and their annotations. ( Human pathways were clustered into SuperPaths based on gene content similarity. GO molecular function: Gene Ontology molecular function. 294 Methylation: is the gene reported to be methylated in cancer by PubMeth? ( 295 GeneRIF: Gene References Into Functions from National Center for Biotechnology Information ( Are any GeneRIF associated with cancer reported for the gene? SLBP: stem-loop binding protein. ECM: extracellular matrix. ERK: extracellular receptor kinase. GTP: guanosine triphosphate. E2F: E2 factor. ErbB: erythroblastosis oncogene B. GPCR: G-protein-coupled receptors. ESC: embryonic stem cells. CREB: camp response element-binding protein.

145 Table 4.8: Summary of search results from PubMed for genes in which somatic variants were identified Gene No. of publications Role of gene Selected references PRMT5 156 PRMT5 is a regulator of homologous recombination-mediated double-strand break repair. PRMT5 methyltransferase activity is necessary for tumour cell proliferation and plays an important role in cancer progression by repressing the expression of key tumour suppressor genes. Mutations in PRMT5 associated with gastric cancer, oropharyngeal squamous cell carcinoma, hepatocellular carcinoma, prostate cancer, lung adenocarcinoma, lung squamous cell carcinoma, endometrial carcinoma and breast carcinoma. ASPN 33 ASPN is a secreted small leucine rich proteoglycan with known roles in ligament regulation and chondrogenesis. It is a potential mediator of metastatic progression found within the tumour microenvironment. ASPN has been shown to play a role in breast cancer, scirrhous gastric cancer, pancreas, and prostate cancer , 404, LAMA2 22 LAMA2 functionally involved in the formation of extracellular matrix and is found to be upregulated in metastatic renal cell carcinoma and during serum-induced glioma initiating cells differentiation. Downregulation of LAMA2 reported in oesophageal cancer, extracellular matrix in drug-resistant ovarian cancer cell line, hepatocellular carcinoma, and laryngeal cancer. Abnormal methylation reported in breast cancer carcinoma and colorectal cancer. LAMA2 is a candidate marker and indicator of poor prognosis for posterior fossa subgroup A epdendymal tumours

146 Gene No. of publications Role of gene Selected references TET2 664 TET2 is an epigenetic regulator which is frequently mutated or inactivated in cancer, and it has been suggested that the TET proteins may protect against abnormal DNA methylation at promoters. TET2 mutations frequently observed in myeloid, lymphoid and hematological malignancies FHOD3 11 FHOD3 involved in cancer cell migration and invasion via regulation of dynamic actin spike assembly in cells invading in vitro and in vivo. FHOD3 plays a role in glioma linear migration motility. FHOD3 was hypomethylated, overexpressed and involved in major deletions and may play a role in thyroid cancer. FHOD3 mutations in leukaemia associated with methotrexate polyglutamates accumulation. GATAD2A 5 GATAD2A is a subunit of the nucleosome remodeling and histone deacetylase complex, a chromatin-level regulator of transcription with a number of important and emerging roles in cancer biology. Knockdown of GATAD2A decreased the ability of cell proliferation and colony formation and promoted cell apoptosis in thyroid cancer cells. A variant in GATAD2A associated with susceptibility to three cancers (breast, ovarian and prostate). ADSSL P4HTM 1 P4HTM found to be hypermethylated in rhabdomyosarcoma. P4HTM silencing by promoter DNA methylation is a potential mechanism for HIF 1α stabilisation in rhabdomyosarcomas. SLC22A20 3 SLC22A20 (OAT6 ) as an uptake carrier of sorafenib, SLC22A20 is differentially methylated in hepatocellular carcinoma , 442, 443

147 Gene No. of publications Role of gene Selected references POLA2 8 POLA2 has been reported to be involved in cell proliferation by mediating DNA replication, recombination, and repair. A variant in POLA2 improves differential survivability and mortality in non-small cell lung cancer patients and could be used as a prognostic biomarker. The knockdown of POLA2 increases gemcitabine resistance in human lung cancer cells. Low mrna expression of POLA2 was prognostic of poor outcome in ovarian carcinomas. POLA2-CDC42EP2 read-through fusion transcript identified in gastrointestinal stromal tumours. POLA2 found to be overexpressed in mesothelioma. 322, ABL1 1,368 The product of the ABL1 gene is a tyrosine kinase which plays a role in cellular growth control and response to DNA damage. The BCR-ABL1 (Philadelphia chromosome) gene fusion is responsible for > 95% of chronic myeloid leukemia. Mutations in BCR-ABL1 gene have been found to be a major cause of disease progression and resistance to tyrosine kinase inhibitors in chronic myeloid leukemia patients. Methylation of the proximal promoter of the ABL1 oncogene is a common epigenetic alteration associated with clinical progression of chronic myeloid leukemia. ABL1 first identified as oncogene in leukaemia but mutations also reported in lung cancer. SDR16C6P PENK 99 PENK is a candidate tumour suppressor gene that is hypermethylated in various cancers. PENK is also a potential biomarker for prostate, colorectal and bladder cancer. Hypermethylation of PENK contributes to cell motility and adhesion. SLC6A18 1 Gain of 5p15.33 (harbouring the SLC6A18 gene) reported in non-small cell lung cancer cases

148 Gene No. of publications Role of gene Selected references PLK2 99 PLK2 plays a critical role in cell cycle and response to DNA damage. PLK2 plays a tumour suppressor role in cervical cancer, ovarian cancer, gastric cancer and hematopoietic diseases. PLK2 is involved in paclitaxel resistance in solid tumours. PLK2 phosphorylates TAp73 resulting in inhibited cell proliferation, increased apoptosis, G1 phase arrest, and decreased cell invasion. Protein kinases represent the most effective class of therapeutic targets in cancer PubMed search was performed using a string ( gene name ) AND (cancer OR malignancy OR tumor* OR tumour* OR sarcoma) in April Abstracts were screened for relevance to the current study.

149 Drug sensitivity Two of the genes identified as of interest (TET2 and LAMA2 ) were reported in the Genomics of Drug Sensitivity in Cancer database. 391 The TET2 gene showed a statistically significant association (p-value < 10 3 ) with VNLG/124 and Bexarotene in pan-cancer analysis (drug sensitivity for cell lines from all cancer types with genomic features identified from the analysis of patient tumours across multiple different cancer types). 391 While the LAMA2 gene was reported in the Genomics of Drug Sensitivity in Cancer database, none of the associations reached statistical significance. VNLG/124 is a novel mutual prodrug of all-trans-retinoic-acid (ATRA) and histone deacetylation inhibitors (HDIs). 475 TET2 mutations determine sensitivity to ATRA. ATRA has been previously shown to induce the interaction and chromatin recruitment of a novel RARβ-TET2 complex to epigenetically activate a specific cohort of target genes. 476 Wu et al. (2017) reported a novel RARβ-TET2-miR-200c-PKCζ signalling pathway that directs cancer cell state changes that may have potential therapeutic implications. 476 Bexarotene is a selective retinoid X receptors (RXR) agonist with properties overlapping ATRA. 477 Bexarotene exerts its effects in blocking cell cycle progression, inducing apoptosis and differentiation, preventing multidrug resistance, and inhibiting angiogenesis and metastasis. 477 Therefore it is a promising chemopreventive agent against cancer. 477 None of the remaining genes harbouring significant somatic mutations were listed in the Genomics of Drug Sensitivity in Cancer database as of April However, as the understanding of genes and pathways that are causally implicated in cancer grows, more therapeutics will be added to the database in the future Loss of heterozygosity variants A total of 2,075 LOH variants were identified in Patient 1-II-2 using VarScan2. Of these, 507 were high confidence. After correcting for multiple testing and removing variants in polygenic regions, 18 LOH variants were statistically significant (Table 4.9). Of these LOH variants, 16 were located on chromosome 16, and the remaining two were located on chromosome

150 There were 1,344 LOH variants identified in Patient 2-II-1 using VarScan2, with 785 categorised as high confidence. After correcting for multiple testing, no LOH variants reached statistical significance for Patient 2-II Copy number analysis The results of DNAcopy were visualised as SCNA graphs per chromosome for Patient 1-II-2 (Appendix I) and Patient 2-II-1 (Appendix J). The SCNA graphs for Patient 1-II-1 show a considerable disruption on chromosome 16. The SCNA graphs for Patient 2-II-1 do not show any large regions of disruption. 119

151 Table 4.9: Statistically significant high confidence loss of heterozygosity variants for Patient 1-II-2 Chr:Pos Gene Somatic p-value Function SIFT PolyPhen-2 RegulomeDB GERP Ref Alt Read depth (Ref, alt) MAF 1000G chr16: JPH x S.. 5. C T 132, chr16: POLR2C 1.22 x Int.... C T 11, chr16: DNAAF x Int.. 1f. G T 6, chr16: ZNF778, ANKRD x IG T P 3a -2 C T 9, chr16: KLHL x S.. 1f. C T 44, chr16: CMTR x S.... C T 10, chr19: CEP x NS T P 2b 2.25 T G 52,0. chr16: ZFP x S.. 3a. A G 82, chr16: PLCG x S.. 3a. C T 82, chr16: CHD x S.... G A 88, chr16: GALNS 5.52 x S.. 4. C T 1, chr16: JPH x S.... T C 6, chr19: CEP x NS T B C T 34,0. chr16: RBL x Int.. 6. T C 3, chr16: WWP x S.. 4. C T 84, chr16: LPCAT x NS T B G A 68, chr16: PLEKHG x Int.. 1f. G A 7, chr16: ZNF x UTR A G 70, Chr:Pos: Chromosome:Position. SIFT: Sorting Intolerant from Tolerant score. PolyPhen-2: Polymorphism Phenotyping-2. GERP: Genomic Evolutionary Rate Profiling score (a positive GERP score represents a substitution deficit, while a negative GERP score represents a substitution surplus). Ref: reference allele. Alt: alternate allele. MAF 1000G: Minor Allele Frequency in 1000 Genomes Project. S: synonymous. NS: nonsynonymous. Int: intronic. IG: intergenic. UTR3: 3 untranslated region. D: deleterious. T: tolerated. B: benign. P: possibly damaging.

152 4.4 Discussion In summary, ten somatic variants in Patient 1-II-2 and three somatic variants in Patient 2-II-1 were identified by VarScan2 and confirmed by Strelka. VarScan2 also identified a large region of LOH on chromosome 16 in Patient 1-II-2. This LOH region was supported by the SCNA results which also indicated a region of SCNA on chromosome 16. Of the somatic mutations identified, two were listed in the Genomics of Drug Sensitivity in Cancer database, indicating the potential clinical utility of these findings. Of the 13 genes in which somatic mutations were identified, 11 genes have been previously associated with cancer in published literature Comparison of results in the context of published literature on myxoid liposarcoma genetics The majority of myxoid liposarcomas are characterised by the presence of the reciprocal chromosomal translocation t(12;16)(q13;p11). This translocation creates the FUS-DDIT3 chimeric gene. 64 A smaller fraction of myxoid liposarcoma cases harbour a similar variant translocation and gene fusion, the t(12;22)(q13;q12), which fuses the EWSR1 gene to the DDIT3 gene. 478 It is likely that these translocations are the primary genetic event essential for tumour formation. 479 However, in solid tumours, single base substitutions outweigh the number of chromosomal translocations by at least one order of magnitude. 16 Therefore, it is possible that sarcomas with fusion gene drivers may also harbour other driver gene mutations. 479 Myxoid liposarcoma can contain several additional molecular genetic alterations, including TP53, PIK3CA, and TERT mutations, which directly influence tumour cell biology and may be involved in round cell transformation, migration capacity, and differential response to drugs Alterations of the TP53 pathway have 480, 486, 487 also been described in myxoid liposarcoma. One study has previously performed a matched tumour and germline analysis on myxoid liposarcoma tumours. Joseph et al. (2014) performed WES on eight fresh frozen surgically resected myxoid liposarcomas and matched blood samples. 488 A median of 10.8 (range 3 15) somatic mutations per tumour were reported, 121

153 consistent with the findings of this study (ten somatic mutations reported in Patient 1-II-2 and three in Patient 2-II-1). One somatic variant was reported by Joseph et al. in FHOD3 gene (g.chr18: g>t). 488 However, this is a different FHOD3 variant to the variant reported in this study. A PubMed search was performed (May 2017) using a string ( gene name ) AND ( myxoid liposarcoma ) for each of the genes in which somatic mutations were identified. No results were returned for any genes except ABL1. It has previously been suggested that ABL1 may play a role in pre- and post-transcriptional regulatory networks that contribute to sensitivity to trabectedin treatment in myxoid liposarcoma patients. 489 The other genes in which somatic mutations were identified in Patient 1-II-2 and Patient 2-II-1 have not been previously reported in myxoid liposarcomas. A cluster of 16 LOH variants on chromosome 16q was also identified in Patient 1-II-2. The SCNA plots for Patient 1-II-2 also highlight a region of SCNA on chromosome 16 which suggests that this may be the site of a significant genomic disruption in this patient. Of the 1,015 genes in this chromosomal region, 66 genes have previously been associated with cancer. Patient 1-II-2 had a LOH mutation in one of the cancer genes located in the region of LOH on chromosome 16, RBL2, at position chr16: (rs ). The minor allele frequency (MAF) in 1000 Genomes Project European population is Therefore, this is a common variant in the general population. The RegulomeDB score for rs is 6, which indicates there is minimal binding evidence at this position. As this is an intronic variant, we do not know the effect of LOH at this position on the phenotype. All other patients in family 1 are also heterozygous at this position, except Patient 1-I-2 (unaffected) who is a homozygous reference (Figure 4.5). Patient 1-III-1 (Ewing s sarcoma) is also heterozygous at this position in the germline DNA, however, without tumour sample for Patient 1-III-1 it is not possible to determine if this variant also becomes homozygous for the alternate allele in the tumour DNA at this position. 122

154 1-I-1 1-I-2 Key Affected male Affected female 1-II-1 * 1-II-2 Sarcoma 1-II-3 Unaffected male Unaffected female 1-III-1 Sarcoma Patient 1-III-2 Genotype at position in RBL2 gene Proband * Patient selected for tumour-normal analysis Read depth Ref, alt Patient 1-I-1 germline T/C 52,71 Patient 1-I-2 germline T/T 115,0 Patient 1-II-1 germline T/C 42,47 Patient 1-II-2 germline T/C 77,60 Patient 1-II-2 tumour C/C 3,60 Patient 1-II-3 germline T/C 70,46 Patient 1-III-1 germline T/C 55,39 Patient 1-III-2 germline T/C 34,55 Figure 4.5: Pedigree of family 1 indicating genotypes for each patient at chr16: (rs ) in the RBL2 gene 123

155 The Retinoblastoma-Like 2 (RBL2 ) gene, also known as RB2 or p130, is a tumour suppressor gene that has been implicated in endometrial cancer, intraocular melanoma, 496, 497 lung cancer, , 508 nasopharyngeal cancer, neuroblastoma, and retinoblastoma The Retinoblastoma (Rb) protein family plays an important role in regulating other cellular processes, such as terminal differentiation and senescence. 528 Previous studies have also shown that Rb proteins are differentially regulated during adipogenic differentiation of pre-adipocyte cell lines, 529, 530 suggesting that an absence of RB1 or RB2 may promote adipogenesis. 531 Human bone marrow-derived mesenchymal stromal cells (hmscs) are multipotent cells that, under defined conditions, can differentiate into multiple connective tissue cell types, such as adipocytes, osteoblasts, chondrocytes, and myoblasts. 532 Differentiation of hmscs into different lineages involves complex regulation and transcriptional activation or repression of a vast number of genes, and disruption of this regulation can 533, 534 have severe pathological consequences, such as cancer development. A second cancer gene of interest in the region of LOH on chromosome 16 in Patient 1-II-2 is the fused in sarcoma (FUS) gene, although no significant variants were reported by VarScan2 in this gene. The FUS gene is involved in the specific translocation of myxoid liposarcomas (t(12;16)(q13;p11)). 535 This translocation fuses exons 5, 7, or 8 of FUS gene with exon 2 of the DDIT3 gene. The FUS gene, also known as translocated in liposarcomas (TLS), is involved in pre-messenger ribonucleic acid (mrna) splicing and the export of fully processed mrna to the cytoplasm. 536 This protein belongs to the FET family of RNA-binding proteins (consisting of FUS, EWS and TAF15) which have been implicated in cellular processes that include regulation of gene expression, maintenance of genomic integrity and mrna/microrna processing. 537 FET genes are directly involved in deleterious genomic rearrangements, primarily in sarcomas and leukaemia. 538 Given that Patient 1-II-2 was diagnosed with a myxoid liposarcoma (a tumour derived from primitive cells that undergo adipose differentiation), the region identified on chromosome 16 may be significant. Chromosome 16 shows a vast region of LOH that encompasses both the RBL2 and FUS genes, as well as 64 other known cancer genes and numerous SCNA events that may contribute towards tumour pathogenesis in this patient. 124

156 4.4.2 Strengths A strength of the current study is the confirmation of statistically significant somatic variants using a second, independent variant caller. Many cancer sequencing studies have relied on a single calling pipeline to generate candidates. However, there is an imperfect consensus between different callers; therefore the results from a single caller should not be over-interpreted. 539 Each caller algorithm has different weaknesses, and VarScan2 has a tendency to return a very high total number of reported calls, which indicates a low specificity. 540 Ideally, more than one algorithm with different biases may reduce the number of false positives. 539 Therefore, statistically significant somatic variants called by VarScan2 were validated using a second somatic variant caller, Strelka. Of the 14 statistically significant somatic variants called by VarScan2 (11 in Patient 1-II-2 and three in Patient 2-II-1), 13 were also called by Strelka (93%). As these somatic variants have been called by two independent callers, it is less likely that these are false positive results. Despite these somatic variants being called by two independent somatic variant callers, these variants should be validated using Sanger sequencing. However, this was beyond the scope of the current project Limitations The analysis of matched tumour and germline data has several unique challenges including accounting for heterogeneity from subclonal variation and sample impurity. 148, 541, 542 The nature of cancer tissue makes somatic variant calling a challenging task. 540 The tumour DNA for this analysis was extracted from FFPE samples collected > ten years earlier. It is hard to determine the tumour purity and heterogeneity from DNA extracted in this manner as it is impossible to verify whether the block contained a mixture of tumour and adjacent normal tissue, or whether the tumour contained heterogeneous cell populations. Therefore, the tumour purity and effects of heterogeneity could not be taken into account in this analysis but should be considered in future studies. 125

157 Other issues that arise from using FFPE samples for WES are artefacts such as fragmentation and artificial base alterations FFPE samples can be a good resource for discovery of biomarkers in cancer using WES, but fresh frozen tissue is preferred as it minimises the damage to nucleotides. 543 There are also sources of error from mapping and sequencing processes. In general, data generated on the Illumina platform have increased error rates at the end of reads, a tendency towards transversion base call errors, a low INDEL error rate, and systematic sequence-specific errors following inverted repeat sequences and GG motifs The matched tumour and germline comparisons were only performed on two of the five sarcoma cases from the three cancer cluster families described in Chapter 2. A clearer picture of the full somatic mutation burden in the three cancer cluster families could be achieved by performing a matched tumour and germline analysis for all sarcomas and other cancers in these families. However, tumour DNA was not available for these patients Summary In summary, 13 novel somatic mutations were identified in two myxoid liposarcoma patients. Two of the genes in which somatic mutations were identified (FHOD3 and ABL1 ) have been previously associated with myxoid liposarcoma in the literature. A large region of LOH and SCNA on chromosome 16q that includes the genes FUS and RBL2 was reported in Patient 1-II-2, which suggests that this chromosomal region may contribute towards tumour pathogenesis in this patient. The genes in which somatic and LOH variants were identified are candidates for further investigation. Independent experimental validation should be performed to screen additional myxoid liposarcomas for variants in these candidate genes. Further functional studies could be carried out to determine the role of these variants or genes in myxoid liposarcoma pathogenesis. Due to time and budget limitations, these types of functional studies are beyond the scope of this thesis. 126

158 Chapter 5 Aim 4: Variant burden analyses at candidate risk loci in sarcoma cases and healthy ageing controls 5.1 Introduction In genetic studies of complex human disease, like cancers, the validation of candidate risk variants is an important and often rate-limiting step Existing single variant association tests are underpowered for validating rare risk variants unless sample or effect sizes are large. 554, 555 A more robust approach involves combining information across variants in a target region, such as a gene. 556 Burden tests use methods that combine rare and common variants across a gene/target region and compare an aggregate statistic between cases and controls. 272, 557, 558 A simple approach is to summarise the genotype information by counting the number of minor alleles across all variants in the target region. 556 In this chapter the candidate risk loci identified in Chapter 3 and Chapter 4 will be assessed by a case-control variant burden analysis to evaluate the full mutational burden of these regions. 127

159 5.1.1 Variant burden analyses in sarcoma cohorts A case-control rare variant burden analysis has previously been performed using sarcoma cases from the International Sarcoma Kindred Study (ISKS) cohort. 178 Targeted exon sequencing was performed on 72 genes associated with increased cancer risk in 1,162 sarcoma cases (including 966 from the ISKS) and 6,545 Caucasian controls. Ballinger et al. found an excess of pathogenic germline variants (combined odds ratio (OR) = 1.43, 95% confidence interval = , p-value < ) with approximately half of the sarcoma cases found to have putatively pathogenic monogenic and polygenic variation in known and novel cancer genes. 178 This study found a measurable contribution of polygenic effects to sarcoma risk by rare variant burden analysis of cases and controls. 178 A variant burden analysis was also performed using 175 Ewing s sarcoma patients from the International Cancer Genome Consortium (100 patients) and Pediatric Cancer Genome Project (19 patients). 559 Pathogenic and likely pathogenic mutations were found in 13.1% of Ewing s sarcoma cases, which is significantly higher compared to the same genes in the Exome Aggregation Consortium (ExAC) database (53,105 subjects). 559 Brohl et al. found pathogenic mutations were highly enriched for genes involved in DNA damage repair and cancer predisposition syndromes. 559 A table of genes identified by Ballinger et al. and Brohl et al. can be found in Appendix L. 5.2 Methods Study participants Sarcoma cases (561) were selected from the ISKS, 175 described previously in Chapter 2. Briefly, the ISKS was initiated in 2008 and is a global resource for researchers to investigate the hereditary characteristics of sarcoma. 175 Patients with sarcoma were recruited from major sarcoma treatment centres across Australia, France, New Zealand, India, the United States of America (USA), the United Kingdom, and Canada, regardless of their family history of cancer. 175 Individuals with adult-onset sarcoma (> 15 years old) were eligible for the ISKS. 128

160 A total of 1,144 healthy ageing cancer-free controls were selected from the Medical Genome Reference Bank (MGRB) program. 560 The MGRB program is a collaborative project between the New South Wales State Government and the Garvan Institute of Medical Research to sequence healthy, older individuals to create a high quality database that is depleted of damaging genetic variants. 560 The MGRB program utilises participants from an existing cohort, the ASPirin in Reducing Events in the Elderly (ASPREE) Study. 561 The ASPREE Study is an international clinical trial to determine whether daily low-dose aspirin improves the quality of life for 19,000 older people in Australia and the USA Whole genome sequencing Whole genome sequencing (WGS) for ISKS cases and MGRB controls was performed by collaborators at the Garvan Institute for Medical Research. Cases and controls were sequenced at one lane per sample on the Illumina HiSeq X Ten platform using TruSeq Nano chemistry (2 x 150 base pair paired-end reads, > 30X mean depth for all samples). Samples passing FastQC 394 and verifybamid 562 contamination filters were mapped to the 1000 Genomes Project hs37d5 reference 563 with an additional PhiX decoy, and small variants called using the Genome Analysis Toolkit (GATK) 3.7 best practices pipeline. 564 The hs37d5 reference is the hg19-based reference genome employed by the 1000 Genomes Project for Phase 3 analysis. This genome differs from the hg19 genome due to the inclusion of 35 Mb of human sequence that is included as an additional contig (hs37d5). Variants passing variant quality score recalibration (VQSR) tranche thresholds of 99.5% (single nucleotide polymorphisms) and 99.0% (insertions and deletions) were retained to summarise frequencies Genomic regions selected for validation Table 5.1 contains eight target regions that were identified in Chapter 3 (ABCB5, ARHGAP39, BEAN1, C16orf96, KIF2C, PDIA2, UVSSA and ZFP69B). These target regions are genes in which germline risk variants segregating with cancer and age at onset of cancer in three cancer-cluster families were identified using whole exome sequencing (WES). 129

161 Table 5.1: Genomic coordinates for target regions in which germline and somatic risk variants were identified Target region Chromosome Start coordinate End coordinate Identified in Chapter 3 KIF2C 1 45,204,490 45,234,438 ZFP69B 1 40,915,337 40,930,390 UVSSA 4 1,340,104 1,382,837 ABCB5 7 20,654,245 20,797,637 ARHGAP ,753, ,839,888 BEAN ,460,200 66,517,745 C16orf ,605,491 4,651,318 PDIA , ,209 Identified in Chapter 4 P4HTM 3 49,026,341 49,045,581 TET ,066, ,201,960 PLK2 5 57,748,810 57,756,966 SLC6A18 5 1,224,470 1,247,304 LAMA ,203, ,838,710 SDR16C6P,PENK 8 57,286,277 57,359,593 ABL ,588, ,764,062 ASPN 9 95,217,489 95,245,844 SLC22A20,POLA ,980,311 65,066,088 ADSSL ,195, ,214,647 PRMT ,388,733 23,399,661 FHOD ,876,702 34,361,018 GATAD2A 19 19,495,642 19,620,741 Genomic coordinates for each target region (± 1,000 bases) based on human genome 19 (hg19) were obtained from the University of California Santa Cruz (UCSC) Genome Browser (

162 The additional 13 target regions listed in Table 5.1 were identified in Chapter 4; (ABL1, ADSSL1, ASPN, FHOD3, GATAD2A, LAMA2, P4HTM, PLK2, PRMT5, SLC6A18, TET2, two target regions encompassing SDR16C6P and PENK, SLC22A20, and POLA2 ). These target regions are genes in which candidate somatic risk variants were identified by a matched tumour and germline analysis in two myxoid liposarcoma patients. For intergenic variants, both flanking genes were included. Genomic coordinates for each target region were obtained from the University of California Santa Cruz (UCSC) Genome Browser ( 565 using human genome build 19 (hg19) and included 1,000 bases either side of each target region. Frequency summary files for the target regions for both case and controls (in variant call format (*.vcf)) were received and annotated using Annotate Variation 245, 257 (ANNOVAR, version 2015Jun16) and Regulome database (RegulomeDB) Statistical analyses Using the annotation from ANNOVAR, the number of nonsynonymous and deleterious alleles (defined as deleterious in both Sorting Intolerant from Tolerant (SIFT) and Polymorphism Phenotyping-2 (PolyPhen-2)) 266, 267 and normal alleles in each target region were summed in cases and controls. As deleterious alleles were defined as being deleterious in both SIFT and PolyPhen-2, this was a more conservative approach. 566 The number of putative regulatory alleles (defined as those with a RegulomeDB score of 1a, 1b, 1c, 1d, 1e, 1f, 2a, 2b or 2c) and normal alleles in each target region were summed in cases and controls. Table 5.2 shows the classification of scores from RegulomeDB. Odds ratios (ORs) and p-values reported for variant burden analysis were obtained from one-sided Fisher s exact tests performed in R 281 to compare the total burden of deleterious and putative regulatory variants, separately, in cases and controls, a method used previously by Ballinger et al. (2016). 178 Bonferroni adjustment was performed to correct for multiple testing

163 Table 5.2: Classification of Regulome database scores Score 1a Supporting data eqtl + TF binding + matched TF motif + matched DNase Footprint + DNase peak 1b eqtl + TF binding + any motif + DNase Footprint + DNase peak 1c 1d 1e 1f 2a 2b 2c 3a 3b eqtl + TF binding + matched TF motif + DNase peak eqtl + TF binding + any motif + DNase peak eqtl + TF binding + matched TF motif eqtl + TF binding / DNase peak TF binding + matched TF motif + matched DNase Footprint + DNase peak TF binding + any motif + DNase Footprint + DNase peak TF binding + matched TF motif + DNase peak TF binding + any motif + DNase peak TF binding + matched TF motif 4 TF binding + DNase peak 5 TF binding or DNase peak 6 Other eqtl: Expression Quantitative Trait Loci. TF: Transcription Factor. DNase: Deoxyribonuclease. 132

164 5.3 Results Identification of nonsynonymous deleterious variants in the target regions The results of the annotation of the frequency summary files using ANNOVAR and RegulomeDB are summarised in Table 5.3. On average, 1,128 variants were identified in each gene in ISKS cohort and 2,282 in the MGRB cohort. Each gene had an average of five nonsynonymous deleterious variants in the ISKS cohort and six nonsynonymous deleterious variants in the MGRB cohort. The ISKS cohort had an average of 12 putative regulatory variants per gene compared to 11 per gene in the MGRB cohort. Table 5.3: Annotated summary of nonsynonymous deleterious variants and putative regulatory variants in the target regions Total variants Deleterious variants Regulatory variants Target region ISKS MGRB ISKS MGRB ISKS MGRB KIF2C ZFP69B P4HTM TET UVSSA PLK SLC6A LAMA2 6,431 7, ABCB5 2,117 22, ARHGAP39 1,085 1, SDR16C6P,PENK 842 1, ABL1 2,173 2, ASPN

165 Total variants Deleterious variants Regulatory variants Target region ISKS MGRB ISKS MGRB ISKS MGRB SLC22A20,POLA ADSSL PRMT BEAN C16orf PDIA FHOD3 5,215 5, GATAD2A 1,456 1, ISKS: International Sarcoma Kindred Study. MGRB: Medical Genome Reference Bank. Deleterious variants: defined as nonsynonymous variants that are deleterious in both Sorting Intolerant from Tolerant (SIFT) and Polymorphism Phenotyping-2 (PolyPhen-2). Regulatory variants: defined as variants with a Regulome database score < 3. Number of variants corresponds to the number of deleterious or regulatory variants within each target region Statistical analyses Nonsynonymous deleterious variants Table 5.4 shows the number of nonsynonymous deleterious alleles and normal alleles for each target region for cases and controls and the results of Fisher s exact test. The significance level after Bonferroni correction was α < 2.38 x A table containing each nonsynonymous deleterious variant for each target region that was included in the variant burden test is located in Appendix K. 134

166 Table 5.4: Odds ratios, p-values and 95% confidence intervals from Fisher s exact test for target regions for nonsynonymous deleterious variants Target region Chr. Identified as Odds ratio p-value 95% CI KIF2C 1 Germline 0 1. ZFP69B 1 Germline P4HTM 3 Somatic UVSSA 4 Germline TET2 4 Somatic x PLK2 5 Somatic 0 1. SLC6A18 5 Somatic x LAMA2 6 Somatic ABCB5 7 Germline ARHGAP39 8 Germline SDR16C6P,PENK 8 Somatic ABL1 9 Somatic ASPN 9 Somatic SLC22A20,POLA2 11 Somatic ADSSL1 14 Somatic PRMT5 14 Somatic BEAN1 16 Germline 0 1. C16orf96 16 Germline x PDIA2 16 Germline x FHOD3 18 Somatic GATAD2A 19 Somatic Chr: Chromosome. ISKS: International Sarcoma Kindred Study. MGRB: Medical Genome Reference Bank. CI: Confidence interval. Odds ratios, p-values and 95% CI obtained from Fisher s exact test performed in R. 135

167 Four target regions reached statistical significance after correction for multiple testing (C16orf96, PDIA2, SLC6A18 and TET2 ). Of these, C16orf96 and PDIA2 were initially identified as germline variants in three cancer cluster families, and SLC6A18 and TET2 were identified as somatic variants from a matched tumour-germline analysis in two myxoid liposarcoma cases. The odds ratios in Table 5.4 indicate a higher burden of nonsynonymous deleterious alleles in sarcoma cases compared to controls for C16orf96, SLC6A18 and TET2. However, the odds ratio for PDIA2 suggests that controls have a higher burden of variant alleles compared to the sarcoma cases Putative regulatory variants Table 5.5 shows the number of putative regulatory alleles and normal alleles for each target region for cases and controls and the results of Fisher s exact test. The significance level after Bonferroni correction was α < 2.78 x A table containing each putative regulatory variant for each target region that was included in the variant burden test is located in Appendix M. 136

168 Table 5.5: Odds ratios and p-values from Fisher s exact test for target regions for putative regulatory variants Target region Chr. Identified as Odds ratio p-value 95% CI KIF2C 1 Germline ZFP69B 1 Germline... P4HTM 3 Somatic UVSSA 4 Germline x TET2 4 Somatic x PLK2 5 Somatic SLC6A18 5 Somatic LAMA2 6 Somatic x ABCB5 7 Germline x ARHGAP39 8 Germline x SDR16C6P,PENK 8 Somatic ABL1 9 Somatic x ASPN 9 Somatic... SLC22A20,POLA2 11 Somatic x ADSSL1 14 Somatic PRMT5 14 Somatic... BEAN1 16 Germline C16orf96 16 Germline x PDIA2 16 Germline FHOD3 18 Somatic x GATAD2A 19 Somatic Chr: Chromosome. ISKS: International Sarcoma Kindred Study. MGRB: Medical Genome Reference Bank. CI: Confidence interval. Odds ratios, p-values and 95% CI obtained from Fisher s exact test performed in R. 137

169 Nine target regions reached statistical significance after correction for multiple testing (ABCB5, ARHGAP39, C16orf96, UVSSA, ABL1, FHOD3, LAMA2, TET2 and a region encompassing SLC22A20 and POLA2 ). Of these, ABCBC5, ARHGAP39, C16orf96, UVSSA were identified as germline variants in three cancer cluster families, and ABL1, FHOD3, LAMA2, TET2 and a region encompassing SLC22A20 and POLA2 were identified as somatic variants from a matched tumour-germline analysis in two myxoid liposarcoma cases. The odds ratios indicate a higher burden of putative regulatory variants in sarcoma cases compared to controls for ARHGAP39, ABL1 and a region encompassing SLC22A20 and POLA2. However, the odds ratio for ABCB5, C16orf96, UVSSA, FHOD3, LAMA2, and TET2 indicates that controls have a higher burden of variant alleles compared to the sarcoma cases. 5.4 Discussion A total of six target regions of interest (C16orf96, SLC6A18, TET2, ARHGAP39, ABL1 and a region encompassing SLC22A20 and POLA2 ) were found to have a higher burden of nonsynonymous deleterious variants or putative regulatory variants in 561 sarcoma cases compared to 1,144 healthy ageing controls Novel findings This is the first study to report associations between the C16orf96, SLC6A18, ARHGAP39, POLA2 and SLC22A20 genes and sarcoma. None of these genes were reported by Ballinger et al. or Brohl et al. in their variant burden analyses 178, 559 in sarcoma cohorts. C16orf96 is an open reading frame gene on chromosome 16 that is an uncharacterised protein coding gene. The function of C16orf96 is currently unknown, and expression is generally low in cells. In situ hybridisation experiments have shown C16orf96 RNA expression is low in testis and skin only and not present in other tissue types. 567 The function of this gene or any potential role for this gene in cancer pathogenesis has not been established. 138

170 The SLC6A18 gene is a member of the SLC6 specific transporter family. SLC6A18 is involved in the transport of glucose and other sugars, bile salts and organic acids, metal ions and amine compounds. A previous study reported a gain of region 5p15.33 containing SLC6A18 in small cell lung cancers. 463 Copy number variations in SLC618A have also been reported in lung adenocarcinoma. 568 The protein encoded by ARHGAP39 is a binding partner for CNK2 that is a spatial modulator of Rac cycling during spine morphogenesis and signalling by G protein coupled receptors (GPCR). 297 There is no supporting evidence for a role for ARHGAP39 in cancer pathogenesis at this time. SLC22A20 is a member of the solute carrier family that plays a role in inorganic anion exchanger activity. SLC22A20 is differentially methylated in hepatocellular 321, 442, 443 carcinoma and may be used as a biomarker for early detection. The POLA2 gene has been reported to be involved in cell proliferation by mediating DNA replication, recombination, and repair. 444 A variant in POLA2 has been found to improve differential survivability and mortality in non-small cell lung cancer patients and could be used as a prognostic biomarker. 445, 448 Low mrna expression of POLA2 was found to be prognostic of poor outcome in ovarian carcinomas. 446 Additionally, POLA2 was found to be overexpressed in mesothelioma. 449 The role of C6orf96, SLC6A18, ARHGAP39, SLC22A20 and POLA2 in sarcoma pathogenesis remains to be elucidated. The results of this study should prioritise further research on these genes in sarcomas Known cancer genes Both the TET2 and ABL1 genes are known cancer genes listed in the Catalogue of Somatic Mutations in Cancer (COSMIC) cancer gene census. 134 TET2 is reported to be frequently mutated or inactivated in cancer and mutations are commonly observed in myeloid, lymphoid and haematological malignancies TET2 has previously been associated with sarcomas. The loss of TET2 is a 569, 570 characteristic of myeloid sarcomas and may be used as a novel marker. 139

171 ABL1 is a proto-oncogene that encodes a protein tyrosine kinase involved in a variety of cellular processes, including cell division, adhesion, differentiation, and response to stress. 571 This gene is known to be fused to a variety of translocation partner genes in various leukaemias, for example, chronic myelogenous leukaemia (BCR-ABL1 ). 572 ABL kinases may also play a role in solid tumours including breast, colon, lung and kidney carcinomas, and melanoma ABL1 variants have previously been reported in sarcomas. Two patients with chronic myeloid leukaemia and secondary sarcomas (histiocytic sarcoma and segregated extramedullary (nodal) myeloid sarcoma) were found to be positive for the t(9;22) BCR/ABL1 translocation in the sarcoma tumours. 583, 584 This evidence suggests that the lineages may be clonally related. 583, 584 However, there is no evidence of ABL1 variants in sarcoma cases without chronic myeloid leukaemia Clinical implications Three of the regions of interest identified in this study may have clinical implications in the treatment of sarcomas. TET2 is listed in the Genomics of Drug Sensitivity in Cancer database and shows a statistically significant association (p-value < 10 3 ) with VNLG/124 and Bexarotene. 391 There may be myeloid sarcomas among the ISKS cases sequenced in this study that harbour TET2 mutations and may respond to VNLG/124 or Bexarotene. However, there may also be other sarcoma subtypes harbouring TET2 mutations. The role of TET2 in sarcoma subtypes other than myeloid sarcomas and treatment of sarcomas with TET2 variants using VNLG/124 and Bexarotene should be further investigated. ABL1 is associated with trabectedin sensitivity in myxoid liposarcomas. 489 Therefore, there may be an opportunity to treat other sarcoma subtypes that exhibit ABL1 variants with trabectedin. An expanded access program tested trabectedin in patients with incurable soft tissue sarcoma following the progression of disease with standard therapy. 585 Results of the study demonstrated disease control despite a low incidence of objective responses in advanced soft tissue sarcoma patients after failure of standard chemotherapy. 585 The study also found greater clinical benefit rate and longer median overall survival in patients with 140

172 leiomyosarcoma and liposarcoma compared with patients with histopathologic subsets of sarcomas other than leiomyosarcoma and liposarcoma. 585 A second study that evaluated the effectiveness of trabectedin for patients with soft tissue sarcoma also found there may be a benefit in using trabectedin in patients with leiomyosarcoma or liposarcoma who failed standard of care agents. 586 The SCL22A20 gene offers some interest and potential clinical utility as an uptake carrier of sorafenib, a multikinase inhibitor. 442 Sorafenib has been shown 587, 588 to have activity in metastatic soft tissue sarcoma, specifically in leiomyosarcoma Strengths and limitations Classic single-marker association analysis for rare variants are underpowered unless the sample size is extremely large, or the variants have a large effect size. 558, 589 Consequently, burden tests for the analysis of rare genetic variants have been developed that consider their joint effects on complex traits within the same functional unit or genomic region. The burden test makes assumptions that all variants in a region are causal and associated with a trait in the same direction and magnitude of effect. 590 Violation of these assumptions can reduce the power of the test For the variants identified in a genomic region by WES and WGS, like in this study, some variants will have little or no effect on the phenotype, some variants may be protective, and some may be deleterious. The magnitude of the effect of each variant may also vary. For example, rare variants may have a larger effect compared to common variants. Some burden tests, for example, sequence kernel association tests, take violations of these assumptions into consideration. 592 However, as only frequency summary files for each cohort were available, the breach of these assumptions could not be addressed at this time. 141

173 There were also several regions of interest that were identified to have a higher mutational rate in controls compared to cases. PDIA2 was found to have a higher rate of nonsynonymous deleterious variants in controls compared to cases. ABCB5, C16orf96, UVSSA, FHOD3, LAMA2 and TET2 were found to have a higher rate of putative regulatory variants in controls compared to cases. This may be due to the presence of common minor alleles in the general population (see Appendix K for minor allele frequencies (MAF) for each variant) or the presence of variants that are phenotypically neutral. Two of the regions of interest (TET2 and C16orf96 ) were found to have a higher mutational rate of nonsynonymous deleterious variants in cases compared to controls, but a higher mutational rate of putative regulatory variants in controls. This may also be due to the presence of common minor alleles classified as putative regulatory variants (see Appendix M for MAF for each variant). For example, two putative regulatory variants in C16orf96 have a MAF of 0.61 and 1.00 in the general population. Therefore, these may be phenotypically neutral variants. Whereas the nonsynonymous deleterious variants in C16orf96 had MAF < 2%. Likewise, TET2 nonsynonymous variants had MAF < 2% whereas one putative regulatory variant had a MAF of Due to these findings of higher mutational rates in controls compared to cases and contradictory findings between nonsynonymous deleterious variants and putative regulatory variants for C16orf96 and TET2, further studies are required to confirm these gene-level associations Conclusion In conclusion, six target regions that were identified by WES in cancer cluster families and matched tumour and germline analysis of two myxoid liposarcomas have been validated using a large independent case and control cohort. C16orf96, SLC6A18 and TET2 were found to have a higher mutational burden of nonsynonymous deleterious variants in sarcoma cases compared to healthy ageing controls. A higher mutational burden of putative regulatory variants in cases was found in ARHGAP39, ABL1 and a region encompassing SCL22A20 and POLA2. This study reported five novel associations between C6orf96, SLC6A18, ARHGAP39, 142

174 POLA2 and SLC22A20 and sarcoma. Two of these genes, TET2 and ABL1, are known cancer genes and have potential clinical utility as they have been identified to contribute to drug sensitivity in cancers. This study has identified novel risk genes that appear to have a higher mutational burden in sarcoma cases compared to healthy ageing controls and should be prioritised for further research. 143

175 144

176 Chapter 6 Conclusion 6.1 Summary of results Whole exome sequencing (WES) was performed on three mixed cancer cluster families identified by a sarcoma proband from the International Sarcoma Kindred Study (ISKS). The cancer cluster families selected were not defined by known cancer predisposition syndromes and therefore represented an opportunity to identify novel risk variants associated with both sarcoma and cancer risk. The WES data was annotated, filtered and prioritised using three different strategies to identify rare private variants, known rare variants and candidate gene variants. The prioritised variants were then tested for association with cancer phenotypes using Sequential Oligogenic Linkage Analysis Routines (SOLAR). Nominally significant variants were assessed for familial segregation in each cancer cluster family. Eight novel putative germline risk variants were identified to segregate with cancer in the families. Each variant was private to a single family and showed segregation with mixed cancer types. These findings suggest the presence of inherited cancer mutations that may increase the risk for cancer within families. 145

177 Matched tumour and germline analyses were performed on two myxoid liposarcoma cases from the cancer cluster families. VarScan2 and Strelka were used to identify 13 novel statistically significant somatic mutations. A vast region of loss of heterozygosity and somatic copy number alterations on chromosome 16 encompassing the RBL2 and FUS genes was also identified in one of the tumours, which may contribute towards tumour pathogenesis. Target regions in which germline and somatic mutations were identified in the cancer cluster families were validated using variant burden analyses in 561 sarcoma cases and 1,144 healthy ageing controls. Six target regions showed an increased mutational burden of nonsynonymous deleterious variants (C16orf96, SLC6A18 and TET2 ) or putative regulatory variants (ARHGAP39, ABL1 and a region encompassing SLC22A20 and POLA2 ) in sarcoma cases compared to controls. 6.2 Clinical utility of findings Two target regions that were found to have a higher mutational burden in sarcoma cases (TET2 and ABL1 ) are known cancer genes and have potential clinical utility in the treatment of sarcomas as they have both been identified to contribute to drug sensitivity in cancers. Also, the SCL22A20 gene offers potential clinical utility as an uptake carrier of sorafenib. TET2 and ABL1 have been reported to be associated with myeloid sarcomas and secondary sarcomas in patients with chronic myeloid leukaemia, respectively. However, there is no evidence of association with other sarcoma subtypes. The remaining genes identified in this study represent novel candidate risk genes for sarcoma. The POLA2 gene has been reported to be involved in cell proliferation by mediating DNA replication, recombination, and repair. 444 The role of the remaining genes of interest (C16orf96, ARHGAP39 and SLC6A18 ) in cancer pathogenesis remain to be elucidated. As previously observed by Ballinger et al. and consistent with the findings of the current study, there is a burden of clinically relevant genetic variation in sarcoma patients and their families. 178 The results from this study will be returned to the ISKS coordinators and submitted to a central database. The database contains molecular and biological information that has been collected over time 146

178 on the ISKS families and specimens. It is critical to catalogue genetic variants as future studies of these candidates may provide a further understanding of the aetiology of sarcoma or new therapies that target these candidates may be developed. 6.3 Review of methodology The current study was the first to perform WES in mixed cancer cluster families identified by a sarcoma proband. This study is an example of a successful two-phase next generation sequencing family study approach; the application of WES to cancer cluster families with rare cancers followed by larger replication in independent population cohorts. The results of the current study show the utility of this approach in small cancer cluster families to identify novel risk genes for a rare disease, such as sarcoma. The current study was limited in the size of the initial study sample (19 people in three families) and assumptions used for variant filtering, prioritisation and segregation analysis, and the availability of tumour DNA. The validation using variant burden analysis was also limited by the inability to account for risk, neutral and protective alleles. The current state of bioinformatic tools, databases and knowledge of cancer biology underpinned the study design and analyses performed. The WES data generated in this study may be re-analysed in the future as new tools are developed and/or the results may become clinically relevant as knowledge in this field progresses. The validation of findings from WES (both germline in families and tumour-germline comparison in myxoid liposarcomas) does not provide conclusive evidence of an involvement of these genes in sarcoma pathogenesis. Rather, the results of this study should be seen as hypothesis-generating for novel candidate risk genes that should be prioritised for future research. 147

179 6.4 Recommendations for future work The current study has identified novel candidate risk genes for sarcoma by performing WES in a small number of cancer cluster families. The role of these genes in sarcoma pathogenesis has not been elucidated in this study and was beyond the scope of this thesis. These genes, however, become candidates that can be further tested for association in other sarcoma and cancer cohorts and for functional validation studies such as molecular assays to determine expression or interactions, or biological assays in animal models. The two-phase NGS family study approach is gaining momentum in genomics literature as researchers return to family-based study designs to identify rare genetic variants. The current study adds to the growing evidence that this approach can be successfully used to identify novel risk genes for a rare complex disease such as sarcoma, and may be extended to identify novel risk genes for other complex diseases. 148

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268 738 Fredrik Mertens, Christopher D.M. Fletcher, Paola Dal Cin, Ivo De Wever, Nils Mandahl, and Felix Mitelman et al. Cytogenetic analysis of 46 pleomorphic soft tissue sarcomas and correlation with morphologic and clinical features: a report of the CHAMP study group. Genes, Chromosomes and Cancer, 22(1):16 25, Anwar N. Mohamed, Mark M. Zalupski, James R. Ryan, Fred Koppitch, Stanley Balcerzak, Raymond Kempf, and Sandra R. Wolman. Cytogenetic aberrations and DNA ploidy in soft tissue sarcoma: a Southwest Oncology Group Study. Cancer Genetics and Cytogenetics, 99(1):45 53, Guidong Li, Akira Ogose, Hiroyuki Kawashima, Hajime Umezu, Tetsuo Hotta, and Tsuyoshi Tohyama et al. Cytogenetic and real-time quantitative reverse-transcriptase polymerase chain reaction analyses in pleomorphic rhabdomyosarcoma. Cancer Genetics and Cytogenetics, 192(1):1 9, Anthony Gordon, Aidan McManus, John Anderson, Cyril Fisher, Syuiti Abe, and Takayuki Nojima et al. Chromosomal imbalances in pleomorphic rhabdomyosarcomas and identification of the alveolar rhabdomyosarcoma-associated PAX3-FOXO1A fusion gene in one case. Cancer Genetics and Cytogenetics, 140(1):73 77, Josette Derre, Real Lagace, Andre Nicolas, Aline Mairal, Frederic Chibon, and Jean-Michel Coindre et al. Leiomyosarcomas and most malignant fibrous histiocytomas share very similar comparative genomic hybridization imbalances: an analysis of a series of 27 leiomyosarcomas. Laboratory Investigation, 81(2): , Marcelo L. Larramendy, Massimiliano Gentile, Sonia Soloneski, Sakari Knuutila, and Tom Bohling. Does comparative genomic hybridization reveal distinct differences in DNA copy number sequence patterns between leiomyosarcoma and malignant fibrous histiocytoma? Cancer Genetics and Cytogenetics, 187(1):1 11, Ana Carneiro, Princy Francis, Par-Ola Bendahl, Josefin Fernebro, Mans Akerman, and Christopher Fletcher et al. Indistinguishable genomic profiles and shared prognostic markers in undifferentiated pleomorphic sarcoma and 237

269 leiomyosarcoma: different sides of a single coin? 89(6): , Laboratory Investigation, 745 Ching C. Lau, Charles P. Harris, Xin-Yan Lu, Laszlo Perlaky, Sheila Gogineni, and Murali Chintagumpala et al. Frequent amplification and rearrangement of chromosomal bands 6p12-p21 and 17p11.2 in osteosarcoma. Genes, Chromosomes and Cancer, 39(1):11 21, Shamini Selvarajah, Maisa Yoshimoto, Olga Ludkovski, Paul C. Park, Jane Bayani, and Paul Thorner et al. Genomic signatures of chromosomal instability and osteosarcoma progression detected by high resolution array CGH and interphase FISH. Cytogenetic and Genome Research, 122(1):5 15, Jane Bayani, Maria Zielenska, Ajay Pandita, Khaldoun Al-Romaih, Jana Karaskova, and Karen Harrison et al. Spectral karyotyping identifies recurrent complex rearrangements of chromosomes 8, 17, and 20 in osteosarcomas. Genes, Chromosomes and Cancer, 36(1):7 16, Julia A. Bridge, Marilu Nelson, Erin McComb, Michael H. McGuire, Howard Rosenthal, and Gerardo Vergara et al. Cytogenetic findings in 73 osteosarcoma specimens and a review of the literature. Cancer Genetics and Cytogenetics, 95(1):74 87,

270 Appendices 239

271

272 Appendix A World Health Organisation classification of soft tissue tumours and bone tumours SOFT TISSUE TUMOURS Adipocytic tumours Benign Lipoma Lipomatosis Lipomatosis of nerve Lipoblastoma / lipoblastomatosis Angiolipoma Myolipoma of soft tissue Chondroid lipoma Extra-renal angiomyolipoma Extra-adrenal myelolipoma Spindle cell / pleomorphic lipoma Hibernoma 241

273 Intermediate (locally aggressive) Atypical lipomatous tumour / well differentiated liposarcoma Malignant Dedifferentiated liposarcoma Myxoid liposarcoma Pleomorphic liposarcoma Liposarcoma, not otherwise specified Atypical lipomatous tumour (ALT) Adipocytic (lipoma-like) Sclerosing Inflammatory types Dedifferentiated liposarcoma Fibroblastic / myofibroblastic tumours Benign Nodular fasciitis Proliferative fasciitis Proliferative myositis Myositis ossifficans Fibro-osseous pseudotumour of digits Ischemic fasciitis Elastofibroma Fibrous hamartoma of infancy Fibromatosis colli Juvenile hyaline fibromatosis Inclusion body fibromatosis Fibroma of tendon sheath Desmoplastic fibroblastoma Mammary-type myofibroblastoma Calcifying aponeurotic fibroma 242

274 Angiomyofibroblastoma Cellular angiofibroma Nuchal-type fibroma Gardner fibroma Calcifying fibrous tumour Intermediate (locally aggressive) Palmar / plantar fibromatosis Desmoids-type fibromatosis Lipofibromatosis Giant cell fibroblastoma Intermediate (rarely metastasizing) Dermatofibrosarcoma protuberans Fibrosarcomatous dermatofibrosarcoma protuberans Pigmented dermatofibrosarcoma protuberans Solitary fibrous tumour Solitary fibrous tumour, malignant Inflammatory myofibroblastic tumour Low grade myofibroblastic sarcoma Myxoinflammatory fibroblastic sarcoma Atypical myxoinflammatory fibroblastic tumour Infantile fibrosarcoma Malignant Adult fibrosarcoma Myxofibrosarcoma Low-grade fibromyxoid sarcoma Sclerosing epithelioid fibrosarcoma Nodular fasciitis Extrapleural solitary fibrous tumour Low grade fibromyxoid sarcoma (LGFMS) 243

275 Sclerosing epithelioid fibrosarcoma (SEF) So-called fibrohistiocytic tumours Benign Tenosynovial giant cell tumour Localized type Diffuse type Malignant Deep benign fibrous histiocytoma Intermediate (rarely metastasizing) Plexiform fibrohistiocytic tumour Giant cell tumour of soft tissue Tenosynovial giant cell tumour Smooth-muscle tumours Benign Leiomyoma of deep soft tissue Malignant Leiomyosarcoma (excluding skin) Leiomyosarcoma Pericytic (perivascular) tumours Glomus tumour (and variants) Glomangiomatosis Malignant glomus tumour Myopericytoma Myofibroma Myofibromatosis Angioleiomyoma Skeletal-muscle tumours Rhabdomyoma Embryonal rhabdomyosarcoma 244

276 Alveolar rhabdomyosarcoma Pleomorphic rhabdomyosarcoma Spindle cell / Sclerosing rhabdomyosarcoma Alveolar rhabdomyosarcoma (ARMS) Vascular tumours Benign Haemangioma Synovial Venous Arteriovenous haemangioma / malformation Epithelioid haemangioma Angiomatosis Lymphangioma Intermediate (locally aggressive) Kaposiform haemangioendothelioma Intermediate (rarely metastasizing) Retiform haemangioendothelioma Papillary intralymphatic angioendothelioma Composite haemangioendothelioma Pseudomyogenic (epithelioid sarcoma-like) haemangioendothelioma Kapsoi sarcoma Malignant Epithelioid haemangioendothelioma Angiosarcoma of soft tissue Gastrointestinal stromal tumours Benign gastrointestinal stromal tumour Gastrointestinal stromal tumour Gastrointestinal stromal tumour 245

277 Nerve sheath tumours Benign Schwannoma (including variants) Melanotic schwannoma Neurofibroma (including variants) Plexiform neurofibroma Perineurioma Malignant perineurioma Granular cell tumour Dermal nerve sheath myxoma Solitary circumscribed neuroma Ectopic meningioma Nasal glial heterotopia Benign Triton tumour Hybrid nerve sheath tumours Malignant Malignant peripheral nerve sheath tumour Epithelioid malignant nerve sheath tumour Malignant Triton tumour Malignant granular cell tumour Ectomesenchymoma Tumours of uncertain differentiation Benign Acral fibromyxoma Intramuscular myxoma (including cellular variant) Juxta-articular myxoma Deep ( aggressive ) angiomyxoma Pleomorphic hyalinizing angiectatic tumour Ectopic hamartomatous thymoma 246

278 Intermediate (locally aggressive) Haemosiderotic fibrolipomatous tumour Intermediate (rarely metastasizing) Atypical fibroxanthoma Angiomatoid fibrous histiocytoma Ossifying fibromyxoid tumour Ossifying fibromyxoid tumour, malignant Mixed tumour NOS Mixed tumour NOS, malignant Myoepithelioma Myoepithelial carcinoma Phosphaturic mesenchymal tumour Phosphaturic mesenchymal tumour Malignant Synovial sarcoma NOS Synovial sarcoma, spindle cell Synovial sarcoma, biphasic Epithelioid sarcoma Alveolar soft-part sarcoma Clear cell sarcoma of soft tissue Extraskeletal myxoid chondrosarcoma Extraskeletal Ewing sarcoma Desmoplastic small round cell tumour Extra-renal rhabdoid tumour Neoplasms with perivascular epithelioid cell differentiation (PEComa) PEComa NOS, benign PEComa NOS, malignant Intimal sarcoma 247

279 Undifferentiated / unclassified sarcomas Undifferentiated spindle cell sarcoma Undifferentiated pleomorphic sarcoma Undifferentiated round cell sarcoma Undifferentiated epithelioid sarcoma Undifferentiated sarcoma NOS Undifferentiated round cell and spindle cell sarcoma Undifferentiated pleomorphic sarcoma (UPS) TUMOURS OF BONE Chondrogenic tumours Benign Osteochondroma Chondroma Enchondroma Periosteal chondroma Osteochondromyxoma Subungual exostosis Bizarre parosteal osteochondromatous proliferation Synovial chondromatosis Intermediate (locally aggressive) Chondromyxoid fibroma Atypical cartilaginous tumour / chondrosarcoma grade I Intermediate (rarely metastasizing) Chondroblastoma Malignant Chondrosarcoma Grade II, Grade III Dedifferentiated chondrosarcoma Mesenchymal chondrosarcoma 248

280 Clear cell chondrosarcoma Osteochondromyxoma Bizarre parosteal osteochondromatous proliferation Chondrosarcoma (grades I-III) Osteogenic tumours Benign Osteoma Osteoid osteoma Intermediate (locally aggressive) Osteoblastoma Malignant Low-grade central osteosarcoma Conventional osteosarcoma Chondroblastic osteosarcoma Fibroblastic osteosarcoma Osteoblastic osteosarcoma Telangiectatic osteosarcoma Small cell osteosarcoma Secondary osteosarcoma Parosteal osteosarcoma Periosteal osteosarcoma High-grade surface osteosarcoma Osteoclastic giant cell rich tumours Benign Giant cell lesion of the small bones Intermediate locally aggressive Giant cell tumour of bone Malignant Malignancy in giant cell tumour of bone 249

281 Fibrohistiocytic tumours Benign Benign fibrous histiocytoma / non-ossifying fibroma Notochordal tumours Benign Benign notochordal tumour Malignant Chordoma Vascular tumours Benign Haemangioma Intermediate locally aggressive rarely metastasizing Epithelioid hemangioma Malignant Epithelioid hemangioendothelioma Angiosarcoma Reference: Bridge, J. A., et al. WHO classification of tumours of soft tissue and bone. International Agency for Research on Cancer,

282 Appendix B Novel tumour-predisposing genes identified by whole exome sequencing 251

283 252 Cancer Population Patients Genes Citation Additional studies Abestos exposed lung adenocarcinoma Finland 26 cases MRPL1, SDK1, SEMA5B, INPP4A 594 Adenomatous Netherlands, 51 patient from 48 families, NTHL1 595 polyposis and USA negative for APC and MUTYH colorectal mutations carcinomas Atypical gastric Spain Large family, with ATP4A 596 neuroendocrine consanguineous parents and tumour, type 1 5/10 affected children Brain Germany 1 family CASP9 597 Breast Poland, Canada 144 Polish and 51 French-Canadian patients with RECQL family history and/or early onset, negative for founder mutations in BRCA1, BRCA2, CHEK2, NBN and PALB2 China 9 early-onset patients with family history, negative for RECQL BRCA1 and BRCA2 mutations Finland 24 breast cancer patients from 11 families, negative for BRCA1 FANCM , 602 and BRCA2 mutations

284 Cancer Population Patients Genes Citation Additional studies Multiple 89 early-onset breast cancer patients from 47 families RINT1 603 Australia 33 breast cancer patients from FANCC, families, negative for BRCA1 BLM and BRCA2 mutations Multiple 13 families XRCC2 608 Finland 129 female hereditary breast ATM, MYC, 609 and/or ovarian cancer patients, PLAU, up to 989 female controls RAD1, and RRM2B Chondrosarcoma France 2 third-degree affected relatives in a single family EXT2 610 Chronic European 59 chronic lymphocytic ITGB2 611 lymphocytic leukaemia-prone families leukaemia and 173 unrelated chronic lymphocytic leukaemia patients UK 66 chronic lymphocytic POT1 612 leukaemia families Colorectal China 23 early onset colorectal cancer patients from 21 families EIF2AK4 613 Spain 3 patients from a large family FAN

285 254 Cancer Population Patients Genes Citation Additional studies Spain Patients from 29 families, CDKN1B, 615 negative for mutations in known XRCC4, colorectal cancer genes EPHX1, NFKBIZ, SMARCA4, BARD1 Finland 4 patients from a large family, negative for mutations in known colorectal cancer genes RPS Finland 96 patients with family history UACA, 617 of colorectal cancer, negative for SFXN4, mutations in known colorectal TWSG1, cancer genes PSPH, NUDT7, ZNF490, PRSS37, CCDC18, PRADC1, MRPL3, AKR1C4 Taiwan 50 colorectal cancer cases NRAS 618 UK 1,006 early-onset familial CRC MRE11, 619 cases and 1,609 healthy controls POLE2 and POT1

286 Cancer Population Patients Genes Citation Additional studies Netherlands 55 colorectal cancer cases with PTPN a disease onset before 45 years and LRP6 of age Colorectal UK Probands from 15 colorectal POLE, adenomas and adenoma families, negative POLD1 carcinomas for mutations in APC and MUTYH Ashkenazi 2 sisters MCM9 630 Colorectal adenomatous polyposis Germany 102 unrelated individuals MSH2 631 Germany 12 colorectal adenomas from DSC2, 629 seven unrelated patients PIEZO1, ZSWIM7 Colorectal Germany 12 colorectal adenomas DSC2, 632 adenomatous from 7 unrelated patients PIEZO1, polyposis with unexplained sporadic ZSWIM7 adenomatous polyposis Esophageal adenocarcinoma and Barrett esophagus USA Large family VSIG10L

287 256 Cancer Population Patients Genes Citation Additional studies Esophageal China 51 stage I and 53 stage III FAM84B 634 squamous cell esophageal squamous cell carcinoma carcinomas Familial China Large family, negative COQ6 635 schwannomatosis for mutations in known disease-causing genes Gastric Finland Large family with the diffuse type of gastric cancer, negative for mutations in CDH1 INSR, FBXO24, DOT1L 636 Netherlands Large family with the diffuse type of gastric cancer, negative for mutations in CDH1 CTNNA Glioma Multiple 90 patients from 55 families POT1 639 Hodgkin Middle East Family with 3 out of 5 affected ACAN 640 lymphoma children and healthy parents Finland Large family with nodular NPAT 641 lymphocyte predominant Hodgkin lymphoma USA 17 Hodgkin lymphoma prone families with three or more KDR 642 affected cases or obligate carriers (69 individuals) Infantile Brazil 2 affected brothers and their NDRG4 643 myofibromatosis healthy consanguineous parents

288 Cancer Population Patients Genes Citation Additional studies Multiple 11 patients from 4 families, and 5 simplex cases PDGFRB USA 11 patients from 9 families PDGFRB 645 USA Large family, negative for PDGFRB mutations NOTCH Invasive pituitary China 6 invasive pituitary adenomas DPCR1, 646 adenomas and 6 non-invasive pituitary EGFL7, adenomas the PRDM family and LRRC50 Juvenile USA Single patient, with extensive SMAD9 647 hamartomatous family history, negative polyposis for known disease-causing syndrome mutations Kaposi sarcoma Finland Large family STAT4 648 Kaposiform Japan Matched tumour and normal ITGB2, 649 hemangioendothelioma sample from an individual IL-32 and DIDO1 Liver France 2 individuals from a family with recurrent well-differentiated hepatocellular tumours DICER1 650 Lung USA Large family PARK Taiwan Large family YAP1 652

289 258 Cancer Population Patients Genes Citation Additional studies Arab An individual with lung cancer from an extended family segregating different types of hereditary cancer NBN 653 Lymphoblastic leukaemia Multiple Large family ETV Male breast Italy 1 male and 2 female BRCA1/2 mutation-negative breast cancer cases from a family Melanoma Multiple 101 patient from 56 melanoma families, negative for CDKN2A and CDK4 mutations Multiple 184 patients from 105 melanoma families, negative for CDKN2A and CDK4 mutations PALB2 656 POT POT USA, Australia, UK Patient from large melanoma family MITF , 660 USA Uveal melanoma patients BAP Finland 21 cases BAP1 594 Melanotic neuroectodermal tumour of infancy UK Single patient CDKN2A 677

290 Cancer Population Patients Genes Citation Additional studies Multiple spinal meningiomas UK 3 unrelated individuals with familial multiple spinal SMARCE meningiomas, negative for mutations in NF2 and SMARCB1 Nasopharyngeal China 161 NPC cases and 895 controls MST1R 682 carcinoma of Southern Chinese descent Nonmedullary thyroid cancer USA Large family HABP2 683 China 5 subjects from a large family RTFC 684 Ovarian UK, USA, 412 high grade serous ovarian FANCM 685 Australia, cancer Germany Paediatric hepatocellular carcinoma USA Single patient ABCB Paediatric poorly differentiated USA Patient with pediatric poorly differentiated carcinoma APC 687 carcinoma Pancreatic ductal Japan 4 cases of KRAS DCTN1-ALK 688 adenocarcinoma mutation-negative pancreatic fusion ductal adenocarcinoma 259 Papillary thyroid carcinoma USA, Canada Large family SRRM2 689

291 260 Cancer Population Patients Genes Citation Additional studies Paraganglioma Spain Patient with multiple paragangliomas and family history of the disease MDH2 690 Penile squamous cell carcinoma UK 27 patients CSN1 691 Pheochromocytoma Spain 3 patients with familial pheochromocytoma, negative for mutations in known disease causing genes MAX , 694 Pre-B cell acute lymphoblastic leukemia Puerto Rican African American ancestry 2 families PAX2 695 Primary thyroid USA 14 female research participants PARP4 696 and breast with primary thyroid and breast cancers without mutations in PTEN

292 Cancer Population Patients Genes Citation Additional studies Primary lymphedema European and Chinese 2 unrelated patients with family history and 1 sporadic case GATA associated with descent a predisposition to acute myeloid leukemia (Emberger syndrome) Prostate USA 91 patient from 19 families BTNL2 705 African American 652 aggressive prostate cancer patients and 752 disease-free controls TET2 706 Japan 140 patients with PC from 66 families TRRAP 707 USA 75 high risk families TANGO2, OR5H14, and CHAD

293 262 Cancer Population Patients Genes Citation Additional studies Australia 5 prostate cancer-affected men PCTP, 709 from 3 families MCRS1, ATRIP, PARP2, CYP3A43, DOK3, PLEKHH3, HEATR5B, GPR124, and HKR1 Renal angiomyolipoma Renal cell carcinoma USA 15 patients TSC1 and TSC2 Korea 10 patients FOXC2 and CLIP Rosette-forming African A patient with rosette-forming FGFR1, 712 glioneuronal American glioneuronal tumour of the PIK3CA, tumour fourth ventricle PTPN11 Small cell carcinoma of the ovary, hypercalcemic type USA, Canada, UK 6 patients from 3 families SMARCA Multiple 7 patients SMARCA , 715, 716 Small intestinal carcinoids USA Large family IPMK 717

294 Cancer Population Patients Genes Citation Additional studies Squamous cell Korea 18 cisplatin-resistant metastatic REV3L 718 carcinoma of head tumours and matched germline and neck Transitional cell carcinoma China 2 patients HECW1 719 Wilms tumour UK 35 families CTR9 720 PubMed search was performed using a string (exome OR exom* OR NGS OR whole genome OR next-generation OR next generation OR WES) AND (familial OR hereditary OR susceptib* OR risk OR germline OR germline ) AND (sequencing OR analysis) AND (cancer OR malignancy OR tumor* OR tumour*) AND English [lang]. Only studies which reported the identification of novel genes by exome sequencing were included. Search results included up to March

295 264

296 Appendix C Familial cancer syndromes associated with sarcomas 265

297 266 Syndrome Sarcoma Inheritance Gene (location) Features Beckwith-Wiedemann syndrome RMS AD NSD1 (5q35.3), KIP2 (11p15.4),CDKN1C (11p15.4), H19 (11p15.5), KCNQ1OT1 (11p15.5), ICR1 (11p15.5) Overgrowth syndrome: macroglossia, omphalocele, hemihypertrophy, gigantism, and associated tumour predisposition Bloom syndrome RMS AR BLM (15q26.1) Progerioid syndrome: growth retardation, sun sensitivity, telangiectasias and other skin changes, and associated tumour predisposition Costello syndrome RMS AD HRAS (11p15.5) Rasopathy: coarse facies, short stature, distinctive hand posture and appearance, cardiac anomalies, developmental delay, congenital myopathy Familial adenomatous polyposis Gardner fibroma, desmoid, RMS AD APC (5q21-q22) Individuals develop hundreds to thousands of polyps of the colon and rectum that can progress to colorectal carcinoma if not treated Familial Gastrointesinal AD KIT (4q12), Multiple gastrointestinal stromal tumours gastrointestinal stromal tumour PDGFRA (4q12) stromal tumour Glomus tumours Glomus tumour AD GLMN (1p22.1) Glomuvenous malformations, glomangioma

298 Syndrome Sarcoma Inheritance Gene (location) Features Gorlin-Goltz nevoid basal cell carcinoma Hereditary leiomyomatosis and renal cell carcinoma syndrome Hereditary Retinoblastoma RMS, fetal rhabdomyoma Leiomyosarcoma (uterus) Sarcomas as second malignant neoplasm, lipoma AD PTCH (Xp11.23) Multiple basal cell carcinomas, odontogenic keratocysts, palmar/plantar pits, calcification of the falx cerebri, rib abnormalities AD FH (1q43) Tumour predisposition syndrome: cutaneous piloleiomyomas, uterine leiomyomas, type 2 papillary renal cell carcinomas AD RB1 (13q14.2) Retinoblastoma, often bilateral and typically in very early childhood Leiomyomatosis-Alport syndrome Leiomyoma XLD COL4A6 (Xq22.3) Alport syndrome plus multiple, diffuse leiomyomas Li-Fraumeni syndrome RMS, undifferentiated pleomorphic sarcoma, pleomorphic liposarcoma AD TP53 (17p13.1) Inherited cancer syndrome: early onset of tumours, multiple tumours within individual; most commonly sarcomas, others include breast cancer, central nervous system tumours, leukaemia and adrenocortical carcinoma Maffucci syndrome Spindle cell IDH1 (2q34), Multiple enchondromas (increased risk of hemangiomas IDH2 (15q26.1) chondrosarcoma) and hemangiomas Mazabraud syndrome Myxomas GNAS1 (20q13.32) Myxomas and fibrous dysplasia 267

299 268 Syndrome Sarcoma Inheritance Gene (location) Features Mosaic variegated aneuploidy RMS AR BUB1B (15q15) Intrauterine growth restriction, microcephaly, spectrum of other anomalies, and a high risk of malignancy including RMS, Wilms, and hematologic malignancy Neurofibromatosis type 1 Neurofibromatosis type 2 Malignant peripheral nerve sheath tumour, RMS, neurofibroma, gastrointestinal stromal tumour Schwannoma, RMS, malignant rhabdoid tumour AD NF1 ( 17q11.2) Cafe-au-lait spots, Lisch nodules in the eye, increased susceptibility to benign and malignant tumours AD NF2 (22q12.2) Tumours of the eighth cranial nerve (usually bilateral) and other schwannomas, meningiomas of the brain, and schwannomas of the dorsal roots of the spinal cord Nijmegen breakage syndrome RMS AR NBS1 (8q21.3) Chromosomal instability syndrome - microcephaly, growth retardation, immunodeficiency, and tumour predisposition Noonan syndrome RMS, lymphangioma AD PTPN11 (12q24) Rasopathy - Dysmorphic facies, short stature, neck webbing, cardiac anomalies, deafness, bleeding diathesis Roberts syndrome RMS AR ESC02 (8p21.1) Range of mild to severe malformation of bones, arms, legs, skull, and face - features similar to those seen in thalidomide exposure

300 Syndrome Sarcoma Inheritance Gene (location) Features Rothmund-Thomson syndrome Osteosarcoma AR RTS (18q24.3) Skin atrophy, telangiectasia, hyper- and hypopigmentation, congenital skeletal abnormalities, short stature, premature ageing, and increased risk of malignant disease Rubinstein-Taybi syndrome RMS AD CREBBP (16p13.1) Multiple congenital anomalies, developmental delay,microcephaly, dysmorphic features, and tumour predisposition Simpson-Golabi-Behmel syndrome Embryonal tumours XLR GPC3 Overgrowth syndrome - coarse facies, congenital heart defects, overgrowth, and other anomalies Tuberous sclerosis RMS, cardiac AD TSC1 (9q34), Hamartomas of multiple organs, rhabdomyoma,chordoma, TSC2 (16p13.3), angiomyolipomas, other renal tumours renal TSC3 (12q22- (cysts and renal cell carcinomas), angiomyolipoma, 24.1) lymphangioleiomyomatosis, angiofibromas perivascular and other skin lesions epithelioid cell tumours Werner syndrome RMS AR WRN (8p12-p11.2) Progerioid syndrome - Scleroderma-like skin changes, early onset atherosclerosis and diabetes AD: autosomal dominant. AR: autosomal recessive. RMS: rhabdomyosarcoma. XLD: X linked dominant. XLR: X linked recessive. 269

301 270

302 Appendix D Translocations associated with sarcomas Translocation Genes Alveolar rhabdomyosarcoma t(2;13)(q36;q14) PAX3 FOXO1 t(1;13)(p36;q14) PAX7 FOXO1 t(8;13;9)(p11;q14;q32) FOXO1-FGFR1 t(x;2)(q13;q36) PAX3-FOXO4 t(2;2)(p23;q36) PAX3-NCOA1 t(2;8)(q36;q13) PAX3-NCOA2 Alveolar soft-part sarcoma t(x;17)(p11.2;q25) ASPL TFE3 Angiomatoid fibrous histiocytoma t(2;22)(q33;q12) EWSR1-CREB1 t(12;16)(q13;p11) FUS-ATF1 t(12;22)(q13;q12) EWSR1-ATF1 Chondroid lipoma t(11;16)(q13.p13) C11orf95-MKL2 271

303 Translocation Genes Clear-cell sarcoma t(2;22)(q33;q12) EWSR1-CREB1 t(12;22)(q13;q12) EWSR1 ATF1 Congenital fibrosarcoma t(12;15)(p13;q25) ETV6 NTRK3 Dedifferentiated liposarcoma t(5;5)(p15;p15) TRIO-TERT t(9;12)(q33;q15) CNOT2-ASTN2?t(12)(q14q14) CTDSP2-FAM19A2 t(9;12)(q33;q21) NR6A1-TRHDE?t(12)(q15q21) NUP107-LGR5 t(9;12)(q33;q15) NUP107-PAPPA t(5;14)(p13;q32) RCOR1-WDR70 Dermatofibrosarcoma protuberans t(17;22)(q22;q13) COL1A1 PDGFB Desmoplastic small round-cell tumour t(11;22)(p13;q12) EWSR1 WT1 t(21;22)(q22;q12) EWSR1-ERG Endometrial stromal sarcoma t(6;10)(p21;p11) EPC1-PHF1 t(6;7)(p21;p15) JAZF1-PHF1 t(7;17)(p15;q11) JAZF1-SUZ12 t(1;6)(p34;p21) MEAF6-PHF1 t(10;17)(q23;p13) YWHAE-FAM22A t(10;17)(q22;p13) YWHAE-FAM22B t(x;22)(p11;q13) ZC3H7B-BCOR Epithelioid hemangioendothelioma t(1;3)(p36;q25) WWTR1-CAMTA1 t(x;11)(p11;q22) YAP1-TFE3 272

304 Translocation Genes Epithelioid sarcoma of the ovary t(12;12)(q23;q24) CMKLR1-HNF1A t(12;12)(q13;q22) ERBB3-CRADD t(1;22)(p36;q11) SMARCB1-WASF2 Ewing s sarcoma t(11;22)(q24;q12) EWSR1 FLI1 t(21;22)(q22;q12) EWSR1 ERG t(7;22)(p22;q12) EWSR1-ER81 t(17;22)(q21;q12) EWSR1-ETV4 t(2;22)(q33;q12) EWSR1 FEV t(21,22)(q22;q12) EWSR1-ERG t(16,21)(p11;q24) FUS-ERG t(2,16)(q35;p11) FUS-FEV t(20,22)(q13;q12) EWSR1-NFATC1 t(6,22)(p21;q12) EWSR1-POU5F1 t(4,22)(q31;q12) EWSR1-SMARCA5 t(7;22)(p21;q12) EWSR1-ETV1 Fibromyxoid sarcoma t(7;16)(q34;p11) FUS-CREB3L2 t(11;16)(p11;p11) FUS-CREB3L1 t(11:22)(p11;q12) EWSR1-CREB3L1 Inflammatory myofibroblastic tumour 2p23 rearrangements TMP3 ALK; TMP4 ALK inv(2)(p23q35) ATIC-ALK t(2;11)(p23;p15) CARS-ALK t(2;17)(p23;q23) CLTC-ALK t(2;12)(p23;p11) PPFIBP1-ALK t(2;2)(p23;q13) RANBP2-ALK t(x;6)(p11;p24) RREB1-TFE3 t(2;4)(p23;q21) SEC31A-ALK t(1;2)(q21;o23) TPM3-ALK 273

305 Translocation Genes t(2;19)(p23;p13) TPM4-ALK t(2;2)(p21;p23) EML4-ALK Kaposi s sarcoma EZH2, SIRT1 Leiomyoma of the uterus inv(7)(p21q22) CUX1-AGR3 t(12;14)(q14;q11) HMGA2-CCNB1IP1 t(7;12)(q31;q14) HMGA2-COG5 t(8;12)(q22;q14) HMGA2-COX6C t(12;14)(q14;q24) HMGA2-RAD51L1 Leiomyosarcoma SIRT1 Lipoblastoma t(7;8)(q21;q12) COL1A2-PLAG1 t(2;8)(q31;q12.1) COL3A1-PLAG1 del(8)(q12q24) HAS2-PLAG1 Lipoma t(5;12)(q33;q14) EBF1-LOC t(2;12)9)(q37;q14) HMGA2-CXCR7 t(5;12)(q33;q14) HMGA2-EBF1 t(12;13)(q14;q13) HMGA2-LHFP t(3;12)(q28;q14 HMGA2-LPP t(9;12)(p22;q14) HMGA2-NFIB t(1;12)(p32;q14) HMGA2-PPAP2B t(3;12)(q28;q14) LPP-C12orf9 Mesenchymal chondrosarcoma t(8;8)(q12;q21) HEY1-NCOA2 t(1;5)(q42;q32) IRFBP2-CDX1 Myoepithelioma t(12;22)(q13;q12) EWSR1-ATF1 t(1;22)(q23;q12) EWSR1-PBX1 t(6;22)(p21;q12) EWSR1-POU5F1 t(19;22)(q13;q12) EWSR1-ZNF

306 Translocation Genes Myxoid chondrosarcoma t(9;17)(q31;q12) TAF15-NR4A3 t(3;9)(q12;q31) TFG-NR4A3 t(9;15)(q31;q21) TCF12-NR4A3 t(9;22)(q22-31;q11-12) EWSR1 NR4A3 Myxoid liposarcoma t(12;16)(q13;p11) FUS DDIT3 t(12;22)(q13;q12) EWSR1 DDIT3 Ossifying fibromyxoid tumour t(6;12)(p21;q24) EP400-PHF1 PEComa t(x;1)(p11;p34) SFPG-TFE3 t(14;x)(q24;q12) RAD51B-OPHN1 t(14;x)(q24;p11) RAD51B-RRAGB Pericytoma t(7;12)(p22;q13) ACTB-GLI1 Primary pulmonary myxoid sarcoma t(2;22)(q33;q12) EWSR1-CREB1 Sclerosing epithelioid fibrosarcoma t(7;16)(q34;p11) FUS-CREB3L2 t(11;22)(p11;q12) EWSR1-CREB3L1 t(7;22)(q3;q12) EWSR1-CREB3L2 Soft tissue angiofibroma t(5;8)(p15;q13) AHRR-NCOA2 t(7;8;14)(q11;q13;q31) GTF2I-NCOA2 Soft tissue chondroma t(3;12)(q28;q14) HMGA2-LPP Solitary fibrous tumour inv(12)(q13q13) NAB2-STAT6 275

307 Translocation Spindle cell rhabdomyosarcoma t(6;8)(p21;q13) t(8;11)(q13;p15) t(6;6)(q22;q24) t(6;8)(q22;q13) Synovial sarcoma t(x;18)(p11;q11) Tenosynovial giant cell tumour t(1;2)(p13;q37) Undifferentiated sarcomas inv(x)(p11p11) t(4;19)(q35;q13) t(10;19)(q26;q13) t(6;22)(p21;q12) t(2;22)(q31;q12) Genes SRF-NCOA2 TEAD1-NCOA2 VGLL2-CITED2 VGLL2-NCOA2 SS18-SSX1, SS18-SSX2, SS18-SSX4 COL6A3-CSF1 BCOR-CCNB3 CIC-DUX4 CIC-DUX4L10 EWSR1-POU5F1 EWSR1-SP3 106, 107, Citations: 276

308 Appendix E Genetically complex sarcomas 277

309 278 Sarcoma Genes References Angiosarcoma Amplification: 8q24.21 (MYC), 10p12.33, 5q Chondrosarcoma (types other than extraskeletal myxoid) COL2A1, IDH1, IDH2, TP53, RB1 pathway 726 Embryonal Polysomy: 8, 2, 11, 12, 13 and 20. Monsomy: 10 rhabdomyosarcoma and 15. LOH: 11p15.5 (IGF2, H19, CDKN1C, HOTS). Gains: 12q13 Fibrosarcoma (other than Multiple non-specific numerical and structural congenital) chromosomal abnormalities. Gain: 22q (PDGF-B) Leiomyosarcoma Gains: 1, 5, 6, 8, 15, 16, 17, 19, 20, 22, X. Losses: 1p, 2, 3, 4, 6q, 8, 9, 10p, 11p, 12q, 11q, 13, 16, 17p, 18 19, 22q. Amplifications: 1, 5, 8, 12, 13, 17, 19, 20 Liposarcoma (types other Gains of 1p, 1q21-q32, 2q, 3p, 3q, 5p12-p15, 5q, than myxoid) 6p21, 7p, 7q22, 8q, 10q, 12q12-q24, 13q, 14q, 15q, 17p, 17q, 18p, 18q12, 19p12, 19q13, 20q, 22q, and Xq21-q27. Losses: 1q, 2q, 3p, 4q, 10q, 11q, 12p13, 13q14, 13q21-qter, 14q23-24, 16q22, 17p13, 17q11.2, and 22q ,

310 Sarcoma Genes References Malignant peripheral Gains: 7p21-q36, 7p22, 7q, 8, 8q11-23, 1q25-44, nerve-sheath tumour and 5q Losses: 1p12-13, 1p21, 1p36, 3p21-pter, 9p13-21, 9p22-24, 10, 10p11-15, 11p, 11q21-25, 13q14, 15p, 16/16q24, 17/17p, 17q11-12, 17q21-25, 22, 22p, 22q13, and 22q Ring chromosomes, trisomy 7, and rearrangements of 11p and 12q Breakpoints: 1p, 7p22 (ETV1 ), 11q13-23, 20q13 (SRC ), and 22q11-13 (NF2 ) Myxofibrosarcoma Gains: 19p, 19q. Losses: 1q, 2q, 3p, 4q, 10q, 11q, and 13q (RB1 ). Amplification: 1, 5p, and 20q Extraskeletal Gains: 1q, 2, 8, and 17p11. Losses: 1q, 2, 5, 6, Osteosarcoma 12, 13, 14, 15, 16, 18, 19, 20, 21, and Y Pleomorphic Gains: 1p22-23, 5, 7p, 8, 14, 18/18, 20p, and 22. rhabdomyosarcoma Losses: 2, 3p, 5q32-qter, 6,10q23 (PTEN ), 11, 13, 14, 15q21-q22, 16, 17, 18, 19, and Y , 738, ,

311 280 Sarcoma Genes References Spindle cell/pleomorphic Gains: 1p36-p31, 1q21-q24, 2p, 4p16, 5p, unclassified sarcoma 5q34, 6q, 7p15-p22, 7q21-qter, 17q, 9q, 14q, 16p13, 17q, 19p13, 19q13.11-q13.2, 20q, and 21q. Losses: 1q32.1, 2p25.3, 2q36-q37, 8p23, 9p, 10q21-q23, 11q22, 13q14-q21, 16q11, and 16q23. Amplifications: 1p33-p34, 12q13-q15, 17cen-p11.2, and 17p13-pter Skeletal osteosarcoma Gains and regional amplifications: 1q, 6p21-p12, 8q23-q24, and 17p13-p11.2 (TP53 ). Partial or complete loss: 6q. Rearrangements of chromosomes

312 Appendix F Known cancer predisposition genes Gene Genomic Cancer predisposition location SDHB 1p36.13 Gastrointestinal stromal tumour, paraganglioma, gastric stromal sarcoma, pheochromocytoma MUTYH 1p34.1 Colorectal adenomas, colorectal adenomatous polyposis, gastric cancer (somatic) UROD 1p34.1 Hepatocellular carcinoma MPL 1p34 Familial essential thrombocythemia GBA 1q22 Gaucher disease SDHC 1q23.3 Gastrointestinal stromal tumour, paraganglioma and gastric stromal sarcoma CDC73 1q31.2 Parathyroid carcinoma and adenoma FH 1q43 Leiomyomatosis and renal cell cancer ALK 2p23.2-p23.1 Familial neuroblastoma SOS1 2p22.1 Noonan syndrome 281

313 Gene Genomic Cancer predisposition location MSH2 2p21-p16 Colorectal cancer, hereditary nonpolyposis, type 1 MSH6 2p16.3 Colorectal cancer, hereditary nonpolyposis type 5, endometrial cancer (familial), mismatch repair cancer syndrome TMEM127 2q11.2 Pheochromocytoma ERCC3 2q14.3 Xeroderma pigmentosum, group B ABCB11 2q31.1 Hepatocellular carcinoma DIS3L2 2q37.1 Perlman syndrome VHL 3p25.3 Hemangioblastoma, pheochromocytoma, renal cell carcinoma, von Hippel-Lindau syndrome XPC 3p25.1 Xeroderma pigmentosum, group C BAP1 3p21.1 Tumour predisposition syndrome COL7A1 3p21.31 Dystrophic epidermolysis bullosa MLH1 3p22.2 Colorectal cancer, hereditary nonpolyposis type 2, mismatch repair cancer syndrome ATR 3q23 Familial cutaneous telangiectasia and cancer syndrome GATA2 3q21.3 Acute myeloid leukemia, myelodysplastic syndrome PHOX2B 4p13 Neuroblastoma KIT 4q12 Gastrointestinal stromal tumour, germ cell tumours, acute myeloid leukemia PDGFRA 4q12 Gastrointestinal stromal tumour SDHA 5p15.33 Paragangliomas TERT 5p15.33 Acute myeloid leukemia, melanoma 282

314 Gene Genomic Cancer predisposition location APC 5q22.2 Adenomatous polyposis coli, Brain tumour-polyposis syndrome 2, Colorectal cancer (somatic), Gardner syndrome, gastric cancer (somatic), hepatoblastoma (somatic) ITK 5q33.3 Lymphoproliferative syndrome 1 HFE 6p22.2 Hemochromatosis FANCE 6p21-p22 Acute myeloid leukaemia POLH 6p21.1 Xeroderma pigmentosum, variant type PMS2 7p22.1 Colorectal cancer, hereditary nonpolyposis type 4, mismatch repair cancer syndrome EGFR 7p11.2 Adenocarcinoma of lung, non-small cell lung cancer SBDS 7q11.21 Shwachman-Diamond syndrome SLC25A13 7q21.3 Hepatocellular carcinoma MET 7q31.2 Hepatocellular carcinoma, renal cell carcinoma, osteofibrous dysplasia PRSS1 7q34 Pancreatic cancer WRN 8p12 Werner syndrome NBN 8q21.3 Acute lymphoblastic leukemia, Nijmegen breakage syndrome EXT1 8q24.11 Chondrosarcoma RECQL4 8q24.3 Rothmund-Thomson syndrome DOCK8 9p24.3 Hyper-IgE recurrent infection syndrome, autosomal recessive MTAP 9p21.3 Malignant fibrous histiocytoma CDKN2A 9p21.3 Melanoma, neural system tumour syndrome, orolaryngeal cancer, pancreatic cancer/melanoma syndrome 283

315 Gene Genomic Cancer predisposition location RMRP 9p13.3 Metaphyseal dysplasia without hypotrichosis FANCG 9p13 Acute myeloid leukaemia XPA 9q22.33 Xeroderma pigmentosum, group A FANCC 9q22.32 Fanconi anemia, complementation group C PTCH1 9q22.32 Basal cell carcinoma TGFBR1 9q22.33 Multiple self-healing squamous epithelioma TSC1 9q34.13 Lymphangioleiomyomatosis, tuberous sclerosis-1 RET 10q11.21 Medullary thyroid carcinoma, multiple endocrine neoplasia, pheochromocytoma BMPR1A 10q23.2 Polyposis syndrome PTEN 10q23.31 Cowden syndrome 1, Endometrial carcinoma, malignant melanoma, PTEN hamartoma tumour syndrome, squamous cell carcinoma, head and neck, glioma susceptibility, prostate cancer TNFRSF6 10q23.31 Autoimmune lymphoproliferative syndrome, squamous cell carcinoma, autoimmune lymphoproliferative syndrome SUFU 10q24.32 Medulloblastoma HRAS 11p15.5 Costello syndrome, bladder cancer, thyroid carcinoma FANCF 11p15 Acute myeloid leukaemia WT1 11p13 Mesothelioma, Wilms tumor, type 1 DDB2 11p11.2 Xeroderma pigmentosum, group E, DDB-negative subtype 284

316 Gene Genomic Cancer predisposition location EXT2 11p11.2 Exostoses, multiple, type 2 SDHAF2 11q12.2 Familial paraganglioma MEN1 11q13.1 Adrenal adenoma, angiofibroma, carcinoid tumour of lung, lipoma, multiple endocrine neoplasia 1, parathyroid adenoma ATM 11q22.3 Lymphoma, T-cell prolymphocytic leukemia, breast cancer CBL 11q23.3 Noonan syndrome-like disorder SDHD 11q23.1 Intestinal carcinoid tumours, Cowden syndrome, merkel cell carcinoma, paraganglioma, gastric stromal sarcoma and pheochromocytoma HMBS 11q23.3 Hepatocellular carcinoma CDKN1B 12p13.1 Multiple endocrine neoplasia, type IV CDK4 12q14.1 Melanoma PTPN11 12q24.13 Juvenile myelomonocytic leukemia, Noonan syndrome 1 HNF1A 12q24.2 Familial hepatic adenoma POLE 12q24.33 Colorectal cancer BRCA2 13q13.1 Fanconi anemia, complementation group D1, Wilms tumour, breast cancer (male), breast-ovarian cancer, glioblastoma, medulloblastoma, pancreatic cancer, prostate cancer GJB2 13q12.11 Vohwinkel syndrome RB1 13q14.2 Bladder cancer, osteosarcoma, retinoblastoma, small cell cancer of the lung ERCC5 13q33.1 Xeroderma pigmentosum, group G MAX 14q23.3 Pheochromocytoma 285

317 Gene Genomic Cancer predisposition location SERPINA1 14q32.13 Thyroid cancer DICER1 14q32.13 Pleuropulmonary blastoma, rhabdomyosarcoma BUB1B 15q15.1 Colorectal cancer FAH 15q25.1 Hepatocellular carcinoma BLM 15q26.1 Bloom Syndrome TSC2 16p13.3 Lymphangioleiomyomatosis, tuberous sclerosis-2 ERCC4 16p13.12 Fanconi anemia, complementation group Q, Xeroderma pigmentosum, group F, Cockayne syndrome PALB2 16p12.2 Fanconi anemia, complementation group N, breast cancer, pancreatic cancer CYLD 16q12.1 Brooke-Spiegler syndrome, cylindromatosis, trichoepithelioma CDH1 16q22.1 Endometrial carcinoma, gastric cancer, ovarian carcinoma, breast cancer, prostate cancer FANCA 16q24.3 Fanconi anemia, complementation group A TP53 17p13.1 Adrenal cortical carcinoma, breast cancer, choroid plexus papilloma, colorectal cancer, hepatocellular carcinoma, Li-Fraumeni syndrome, nasopharyngeal carcinoma, osteosarcoma, pancreatic cancer, basal cell carcinoma 7, glioma susceptibility FLCN 17p11.2 Colorectal cancer, renal carcinoma RAD51D 17q12 Familial breast-ovarian cancer NF1 17q11.2 Neurofibromatosis, type 1 286

318 Gene Genomic Cancer predisposition location BRCA1 17q21.31 Familial breast-ovarian cancer, pancreatic cancer STAT3 17q21.2 Autoimmune disease, Hyper-IgE recurrent infection syndrome SMARCE1 17q21.2 Familial meningioma TRIM37 17q22 Breast cancer BRIP1 17q23.2 Breast cancer, Fanconi anemia, complementation group J PRKAR1A 17q24.2 Adrenocortical tumour, Carney complex type 1, pigmented nodular adrenocortical disease, primary AXIN2 17q24.1 Colorectal cancer, oligodontia-colorectal cancer syndrome RAD51C 17q22 Fanconi anemia, complementation group O, familial breast-ovarian cancer RHBDF2 17q25.1 Tylosis with esophageal cancer SMAD4 18q21.2 Juvenile polyposis/hereditary hemorrhagic telangiectasia syndrome, pancreatic cancer (somatic), juvenile intestinal polyposis SETBP1 18q21.1 Schinzel-Giedion syndrome ELANE 19p13.3 Cyclic neutropenia, severe congenital neutropenia STK11 19p13.3 Melanoma, pancreatic cancer, Peutz-Jeghers syndrome, testicular cancer SMARCA4 19p13.2 Coffin-Siris syndrome 4, rhabdoid tumour predisposition syndrome 2 CEBPA 19q13.11 Acute myeloid leukaemia 287

319 Gene Genomic Cancer predisposition location ERCC2 19q13.32 Cerebrooculofacioskeletal syndrome 2, trichothiodystrophy 1 photosensitive, xeroderma pigmentosum group D POLD1 19q13.33 Colorectal cancer RUNX1 21q22.12 Acute myeloid leukaemia, platelet disorder, familial, with associated myeloid malignancy SMARCB1 22q11.23 Coffin-Siris syndrome, Rhabdoid tumours, Schwannomatosis-1 LZTR1 22q11.21 Schwannomatosis-2 CHEK2 22q12.1 Li-Fraumeni syndrome, osteosarcoma, breast cancer, colorectal cancer, prostate cancer NF2 22q12.2 Neurofibromatosis type 2, schwannomatosis, meningioma WAS Xp11.23 Neutropenia, thrombocytopenia, Wiskott-Aldrich syndrome SH2D1A Xq25 Lymphoproliferative syndrome GPC3 Xq26.2 Wilms tumour DKC1 Xq28 Dyskeratosis congenita SRY Yp11.2 Hepatocellular carcinoma The information in this table was sourced from the Online Inheritance in Man database and 134, the Catalogue of Somatic Mutations in Cancer database. 288

320 Appendix G Candidate genes used for variant prioritisation based on a priori knowledge of cancer biology Gene name Chromosome Start End No. variants APC ARID1A ATM ATR AXIN AXIN BARD BLM BRCA BRCA BRIP BUB1B C17orf CD99 Y CDH CDKN2A CHEK CHEK DDB DICER

321 Gene name Chromosome Start End No. variants DKC1 X DNA ELF ELF ERCC ERCC ERCC ERCC ERF ERG ETS ETS ETV ETV ETV ETV EWSR EXT EXT FAM175A FANCA FANCB X FANCC FANCD FANCE FANCF FANCG FANCI FANCL FANCM FH FLI HNF4A IDH IDH KIF1B KIT LIG LIG MDM MEN

322 Gene name Chromosome Start End No. variants MET MLH MLH MRE11A MSH MSH MSH MUTYH NBN NEIL NF NF PALB PMS PMS POLH PPARG PRKAR1A PTCH PTEN PTPN RAD RAD51C RAD51D RB RECQL RET RMI RMI RPA RPA RPS SDHA SDHB SDHC SDHD SMARCA SMARCB SPDEF SPI SQSTM

323 Gene name Chromosome Start End No. variants STK TAF TGFBR TNFRSF11A TOP TOP3A TP TP53BP TSC TSC VHL WRN WT XPA XPC XRCC Start and End: the chromosome locations of the start and end of the gene (including ± 25 kb). 292

324 Appendix H Genes in which variants were also prioritised using the candidate gene prioritisation strategy Gene Chromosome No. variants ACCS 11 8 ACP ACRV ACYP AIMP2 7 3 ALX ANKRD ARHGAP ARHGDIG 16 2 ARHGEF ATP13A2 1 4 ATP1B ATP5D 19 3 BRK1 3 3 C11orf C11orf C19orf C22orf C5orf C9orf

325 Gene Chromosome No. variants CAMKK CAT 11 2 CCDC CDC42BPG CFAP CHCHD COX6B CPM 12 4 DCTN DERL DHFR 5 3 DHX DLAT 11 1 DMRTC EIF2AK1 7 1 EIF2B EPCAM 2 2 EPM2AIP1 3 1 EVI2A 17 2 FAM20A 17 3 FAM20A, PRKAR1A 17 1 FBXO FDFT1 8 4 FLII 17 7 FNDC FRY 13 1 GAS2L GATA4 8 3 GPT 8 2 GSK3A 19 3 GTPBP2 6 2 HAUS HEATR HELQ 4 11 HNRNPK 9 4 HSCB 22 2 IL INTS IRAK2 3 4 ITFG ITGAE

326 Gene Chromosome No. variants KLC LOC LOC LPAR LRRC LRRC14B 5 2 LRRFIP2 3 2 LYPD MAD2L1BP 6 1 MAP3K2 2 1 MAP4K MFSD3 8 1 MIDN 19 3 MIEF MIS18BP MMP MPZ 1 1 MRPL MRPS18C 4 1 MYBPC NDUFAB NPAT 11 1 NR1H ORMDL1 2 3 OSGIN2 8 2 PACSIN1 6 1 PADI2 1 9 PDIA PGD 1 3 PIGO 9 10 PIGV 1 2 PIH1D PKD PLA2G4C PLCG1-AS POLG PPP1R16A 8 2 R3HDML 20 6 RBM RCBTB RGS

327 Gene Chromosome No. variants RHBDD RNPEP 1 5 RNU6-28P 15 5 RPL10A 6 1 RUFY SHMT SLC2A SLC9A3R SMCR SMYD SRP STOML2 9 3 STT3A 11 3 TANGO TEAD3 6 4 TESK2 1 3 TMEM TMEM8A TOE1 1 1 UPK1A 19 3 VAT VCP 9 5 VPS9D VRK2 2 1 WRAP XPO5 6 2 XRN1 3 1 ZAR1L 13 4 ZC2HC1C 14 2 ZNF ZNF ZNF

328 Appendix I Patient 1-II-2: Copy number variation by chromosome 297

329 Log 2 (T/R) Log 2 (T/R) Log 2 (T/R) Log 2 (T/R) Log 2 (T/R) Log 2 (T/R) Index Index Index Index Index Index Black: normalised log ratios. Red: mean values among points in segment obtained by circular binary segmentation.

330 Log 2 (T/R) Log 2 (T/R) Log 2 (T/R) Log 2 (T/R) Log 2 (T/R) Log 2 (T/R) Index Index Index Index Index Index Black: normalised log ratios. Red: mean values among points in segment obtained by circular binary segmentation.

331 Log 2 (T/R) Log 2 (T/R) Log 2 (T/R) Log 2 (T/R) Log 2 (T/R) Log 2 (T/R) Index Index Index Index Index Index Black: normalised log ratios. Red: mean values among points in segment obtained by circular binary segmentation.

332 Log 2 (T/R) Log 2 (T/R) Log 2 (T/R) Log 2 (T/R) Log 2 (T/R) Log 2 (T/R) Index Index Index Index Index Index Black: normalised log ratios. Red: mean values among points in segment obtained by circular binary segmentation.

333

334 Appendix J Patient 2-II-1: Copy number variation by chromosome 303

335 Log 2 (T/R) Log 2 (T/R) Log 2 (T/R) Log 2 (T/R) Log 2 (T/R) Log 2 (T/R) Index Index Index Index Index Index Black: normalised log ratios. Red: mean values among points in segment obtained by circular binary segmentation.

336 Log 2 (T/R) Log 2 (T/R) Log 2 (T/R) Log 2 (T/R) Log 2 (T/R) Log 2 (T/R) Index Index Index Index Index Index Black: normalised log ratios. Red: mean values among points in segment obtained by circular binary segmentation.

337 Log 2 (T/R) Log 2 (T/R) Log 2 (T/R) Log 2 (T/R) Log 2 (T/R) Log 2 (T/R) Index Index Index Index Index Index Black: normalised log ratios. Red: mean values among points in segment obtained by circular binary segmentation.