Andrew Lonie

Whole–genome characterization of chemoresistant ovarian cancer

Patients with high-grade serous ovarian cancer (HGSC) have experienced little improvement in overall survival, and standard treatment has not advanced beyond platinum-based combination chemotherapy, during the past 30 years. To understand the drivers of clinical phenotypes better, here we use whole-genome sequencing of tumour and germline DNA samples from 92 patients with primary refractory, resistant, sensitive and matched acquired resistant disease. We show that gene breakage commonly inactivates the tumour suppressors RB1, NF1, RAD51B and PTEN in HGSC, and contributes to acquired chemotherapy resistance. CCNE1 amplification was common in primary resistant and refractory disease. We observed several molecular events associated with acquired resistance, including multiple independent reversions of germline BRCA1 or BRCA2 mutations in individual patients, loss of BRCA1 promoter methylation, an alteration in molecular subtype, and recurrent promoter fusion associated with overexpression of the drug efflux pump MDR1.

The alpha-1,3-galactosyltransferase knockout mouse. Implications for xenotransplantation.

Organ xenografts in discordant combinations such as pig-to-man undergo hyperacute rejection due to the presence of naturally occurring human anti-pig xenoantibodies. The galactose alpha(1,3)-galactose epitope on glycolipids and glycoproteins is the major porcine xenoantigen recognized by these xenoantibodies. This epitope is formed by alpha(1,3)-galactosyltransferase, which is present in all mammals except man, apes, and Old World monkeys. We have generated mice lacking this major xenoantigen by inactivating the alpha(1,3)-galactosyltransferase gene. These mice are viable and have normal organs but develop cataracts. Substantially less xenoantibody from human serum binds to cells and tissues of these mice compared with normal mice. Similarly, there is less activation of human complement on cells from mice lacking the galactose alpha(1,3)-galactose epitope. These mice confirm the importance of the galactose alpha(1,3)-galactose epitope in human xenoreactivity and the logic of continuing efforts to generate pigs that lack this epitope as a source of donor organs.

Tracking the origins and drivers of subclonal metastatic expansion in prostate cancer

Tumour heterogeneity in primary prostate cancer is a well-established phenomenon. However, how the subclonal diversity of tumours changes during metastasis and progression to lethality is poorly understood. Here we reveal the precise direction of metastatic spread across four lethal prostate cancer patients using whole-genome and ultra-deep targeted sequencing of longitudinally collected primary and metastatic tumours. We find one case of metastatic spread to the surgical bed causing local recurrence, and another case of cross-metastatic site seeding combining with dynamic remoulding of subclonal mixtures in response to therapy. By ultra-deep sequencing end-stage blood, we detect both metastatic and primary tumour clones, even years after removal of the prostate. Analysis of mutations associated with metastasis reveals an enrichment of TP53 mutations, and additional sequencing of metastases from 19 patients demonstrates that acquisition of TP53 mutations is linked with the expansion of subclones with metastatic potential which we can detect in the blood.

Rare Mutations in XRCC2 Increase the Risk of Breast Cancer

An exome-sequencing study of families with multiple breast-cancer-affected individuals identified two families with XRCC2 mutations, one with a protein-truncating mutation and one with a probably deleterious missense mutation. We performed a population-based case-control mutation-screening study that identified six probably pathogenic coding variants in 1,308 cases with early-onset breast cancer and no variants in 1,120 controls (the severity grading was p < 0.02). We also performed additional mutation screening in 689 multiple-case families. We identified ten breast-cancer-affected families with protein-truncating or probably deleterious rare missense variants in XRCC2. Our identification of XRCC2 as a breast cancer susceptibility gene thus increases the proportion of breast cancers that are associated with homologous recombination-DNA-repair dysfunction and Fanconi anemia and could therefore benefit from specific targeted treatments such as PARP (poly ADP ribose polymerase) inhibitors. This study demonstrates the power of massively parallel sequencing for discovering susceptibility genes for common, complex diseases.

Targeting gene expression to endothelium in transgenic animals: a comparison of the human ICAM-2, PECAM-1 and endoglin promoters.

Abstract: It is highly likely that successful pig‐to‐human xenotransplantation of vascularized organs will require genetic modification of the donor pig, and in particular of donor vascular endothelium. Promoters are generally tested in transgenic mice before generating transgenic pigs. Several promoters have been used to drive endothelial cell‐specific expression in mice but none have yet been tested in pigs. We compared the promoters of three human genes that are predominantly expressed in vascular endothelium: intercellular adhesion molecule 2 (ICAM‐2), platelet endothelial cell adhesion molecule 1 (PECAM‐1) and endoglin. Expression of human complement regulatory proteins (hCRPs), directed by each of the promoters in mice, was largely restricted to vascular endothelium and leukocyte subpopulations. However, expression from the PECAM‐1 promoter was weak in liver and non‐uniform in the small vessels of heart, kidney, and lung. Conversely, expression from the endoglin promoter was consistently strong in the small vessels of these organs but was absent in larger vessels. The ICAM‐2 promoter, which produced strong and uniform endothelial expression in all organs examined, was therefore used to generate hCRP transgenic pigs. Leukocytes from 57 pigs containing at least one intact transgene were tested for transgene expression by flow cytometry. Forty‐seven of these transgenic pigs were further analyzed by immunohistochemical staining of liver biopsies, and 18 by staining of heart and kidney sections. Only two of the pigs showed expression, which appeared to be restricted to vascular endothelium in heart and kidney but was markedly weaker than in transgenic mice produced with the same batch of DNA. Thus, in this case, promoter performance in mice and pigs was not equivalent. The weak expression driven by the human ICAM‐2 promoter in pigs relative to mice suggests the need for additional regulatory elements to achieve species‐specific gene expression in pigs.

Genomics Virtual Laboratory: A Practical Bioinformatics Workbench for the Cloud.

Background Analyzing high throughput genomics data is a complex and compute intensive task, generally requiring numerous software tools and large reference data sets, tied together in successive stages of data transformation and visualisation. A computational platform enabling best practice genomics analysis ideally meets a number of requirements, including: a wide range of analysis and visualisation tools, closely linked to large user and reference data sets; workflow platform(s) enabling accessible, reproducible, portable analyses, through a flexible set of interfaces; highly available, scalable computational resources; and flexibility and versatility in the use of these resources to meet demands and expertise of a variety of users. Access to an appropriate computational platform can be a significant barrier to researchers, as establishing such a platform requires a large upfront investment in hardware, experience, and expertise. Results We designed and implemented the Genomics Virtual Laboratory (GVL) as a middleware layer of machine images, cloud management tools, and online services that enable researchers to build arbitrarily sized compute clusters on demand, pre-populated with fully configured bioinformatics tools, reference datasets and workflow and visualisation options. The platform is flexible in that users can conduct analyses through web-based (Galaxy, RStudio, IPython Notebook) or command-line interfaces, and add/remove compute nodes and data resources as required. Best-practice tutorials and protocols provide a path from introductory training to practice. The GVL is available on the OpenStack-based Australian Research Cloud (http://nectar.org.au) and the Amazon Web Services cloud. The principles, implementation and build process are designed to be cloud-agnostic. Conclusions This paper provides a blueprint for the design and implementation of a cloud-based Genomics Virtual Laboratory. We discuss scope, design considerations and technical and logistical constraints, and explore the value added to the research community through the suite of services and resources provided by our implementation.

Cpipe: a shared variant detection pipeline designed for diagnostic settings

The benefits of implementing high throughput sequencing in the clinic are quickly becoming apparent. However, few freely available bioinformatics pipelines have been built from the ground up with clinical genomics in mind. Here we present Cpipe, a pipeline designed specifically for clinical genetic disease diagnostics. Cpipe was developed by the Melbourne Genomics Health Alliance, an Australian initiative to promote common approaches to genomics across healthcare institutions. As such, Cpipe has been designed to provide fast, effective and reproducible analysis, while also being highly flexible and customisable to meet the individual needs of diverse clinical settings. Cpipe is being shared with the clinical sequencing community as an open source project and is available at http://cpipeline.org.

Rare Mutations in RINT1 Predispose Carriers to Breast and Lynch Syndrome–Spectrum Cancers

UNLABELLED Approximately half of the familial aggregation of breast cancer remains unexplained. A multiple-case breast cancer family exome-sequencing study identified three likely pathogenic mutations in RINT1 (NM_021930.4) not present in public sequencing databases: RINT1 c.343C>T (p.Q115X), c.1132_1134del (p.M378del), and c.1207G>T (p.D403Y). On the basis of this finding, a population-based case-control mutation-screening study was conducted that identified 29 carriers of rare (minor allele frequency < 0.5%), likely pathogenic variants: 23 in 1,313 early-onset breast cancer cases and six in 1,123 frequency-matched controls [OR, 3.24; 95% confidence interval (CI), 1.29-8.17; P = 0.013]. RINT1 mutation screening of probands from 798 multiple-case breast cancer families identified four additional carriers of rare genetic variants. Analysis of the incidence of first primary cancers in families of women carrying RINT1 mutations estimated that carriers were at increased risk of Lynch syndrome-spectrum cancers [standardized incidence ratio (SIR), 3.35; 95% CI, 1.7-6.0; P = 0.005], particularly for relatives diagnosed with cancer under the age of 60 years (SIR, 10.9; 95% CI, 4.7-21; P = 0.0003). SIGNIFICANCE The work described in this study adds RINT1 to the growing list of genes in which rare sequence variants are associated with intermediate levels of breast cancer risk. Given that RINT1 is also associated with a spectrum of cancers with mismatch repair defects, these findings have clinical applications and raise interesting biological questions.

Identification of G protein-coupled receptors in Schistosoma haematobium and S. mansoni by comparative genomics

BackgroundSchistosomiasis is a parasitic disease affecting ~200 million people worldwide. Schistosoma haematobium and S. mansoni are two relatively closely related schistosomes (blood flukes), and the causative agents of urogenital and hepatointestinal schistosomiasis, respectively. The availability of genomic, transcriptomic and proteomic data sets for these two schistosomes now provides unprecedented opportunities to explore their biology, host interactions and schistosomiasis at the molecular level. A particularly important group of molecules involved in a range of biological and developmental processes in schistosomes and other parasites are the G protein-coupled receptors (GPCRs). Although GPCRs have been studied in schistosomes, there has been no detailed comparison of these receptors between closely related species. Here, using a genomic-bioinformatic approach, we identified and characterised key GPCRs in S. haematobium and S. mansoni (two closely related species of schistosome).MethodsUsing a Hidden Markov Model (HMM) and Support Vector Machine (SVM)-based pipeline, we classified and sub-classified GPCRs of S. haematobium and S. mansoni, combined with phylogenetic and transcription analyses.ResultsWe identified and classified classes A, B, C and F as well as an unclassified group of GPCRs encoded in the genomes of S. haematobium and S. mansoni. In addition, we characterised ligand-specific subclasses (i.e. amine, peptide, opsin and orphan) within class A (rhodopsin-like).ConclusionsMost GPCRs shared a high degree of similarity and conservation, except for members of a particular clade (designated SmGPR), which appear to have diverged between S. haematobium and S. mansoni and might explain, to some extent, some of the underlying biological differences between these two schistosomes. The present set of annotated GPCRs provides a basis for future functional genomic studies of cellular GPCR-mediated signal transduction and a resource for future drug discovery efforts in schistosomes.

Towards A Systemic Framework for Digital Forensic Readiness

Although digital forensics has traditionally been associated with law enforcement, the impact of new regulations, industry standards and cyber-attacks, combined with a heavy reliance on digital assets, has resulted in a more prominent role for digital forensics in organizations. Modern organizations, therefore, need to be forensically ready in order to maximize their potential to respond to forensic events and demonstrate compliance with laws and regulations. However, little research exists on the assessment of organizational digital forensic readiness. This paper describes a comprehensive approach to identifying the factors that contribute to digital forensic readiness and how these factors work together to achieve forensic readiness in an organization. We develop a conceptual framework for organizational forensic readiness and define future work towards the empirical validation and refinement of the framework.