Publications by authors named "Karin S Kassahn"

35 Publications

Identification and targeted management of a neurodegenerative disorder caused by biallelic mutations in SLC5A6.

NPJ Genom Med 2019 14;4:28. Epub 2019 Nov 14.

1Genetics and Molecular Pathology Research Laboratory, Centre for Cancer Biology, An alliance between SA Pathology and the University of South Australia, Adelaide, SA Australia.

We describe a sibling pair displaying an early infantile-onset, progressive neurodegenerative phenotype, with symptoms of developmental delay and epileptic encephalopathy developing from 12 to 14 months of age. Using whole exome sequencing, compound heterozygous variants were identified in , which encodes the sodium-dependent multivitamin transporter (SMVT) protein. SMVT is an important transporter of the B-group vitamins biotin, pantothenate, and lipoate. The protein is ubiquitously expressed and has major roles in vitamin uptake in the digestive system, as well as transport of these vitamins across the blood-brain barrier. Pathogenicity of the identified variants was demonstrated by impaired biotin uptake of mutant SMVT. Identification of this vitamin transporter as the genetic basis of this disorder guided targeted therapeutic intervention, resulting clinically in improvement of the patient's neurocognitive and neuromotor function. This is the second report of biallelic mutations in leading to a neurodegenerative disorder due to impaired biotin, pantothenate and lipoate uptake. The genetic and phenotypic overlap of these cases confirms mutations in as the genetic cause of this disease phenotype. Recognition of the genetic disorder caused by mutations is essential for early diagnosis and to facilitate timely intervention by triple vitamin (biotin, pantothenate, and lipoate) replacement therapy.
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http://dx.doi.org/10.1038/s41525-019-0103-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6856110PMC
November 2019

Truncating Variants in NAA15 Are Associated with Variable Levels of Intellectual Disability, Autism Spectrum Disorder, and Congenital Anomalies.

Am J Hum Genet 2018 05 12;102(5):985-994. Epub 2018 Apr 12.

Kennedy Krieger Institute, 801 North Broadway Baltimore, MD 21205, USA.

N-alpha-acetylation is a common co-translational protein modification that is essential for normal cell function in humans. We previously identified the genetic basis of an X-linked infantile lethal Mendelian disorder involving a c.109T>C (p.Ser37Pro) missense variant in NAA10, which encodes the catalytic subunit of the N-terminal acetyltransferase A (NatA) complex. The auxiliary subunit of the NatA complex, NAA15, is the dimeric binding partner for NAA10. Through a genotype-first approach with whole-exome or genome sequencing (WES/WGS) and targeted sequencing analysis, we identified and phenotypically characterized 38 individuals from 33 unrelated families with 25 different de novo or inherited, dominantly acting likely gene disrupting (LGD) variants in NAA15. Clinical features of affected individuals with LGD variants in NAA15 include variable levels of intellectual disability, delayed speech and motor milestones, and autism spectrum disorder. Additionally, mild craniofacial dysmorphology, congenital cardiac anomalies, and seizures are present in some subjects. RNA analysis in cell lines from two individuals showed degradation of the transcripts with LGD variants, probably as a result of nonsense-mediated decay. Functional assays in yeast confirmed a deleterious effect for two of the LGD variants in NAA15. Further supporting a mechanism of haploinsufficiency, individuals with copy-number variant (CNV) deletions involving NAA15 and surrounding genes can present with mild intellectual disability, mild dysmorphic features, motor delays, and decreased growth. We propose that defects in NatA-mediated N-terminal acetylation (NTA) lead to variable levels of neurodevelopmental disorders in humans, supporting the importance of the NatA complex in normal human development.
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http://dx.doi.org/10.1016/j.ajhg.2018.03.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5986698PMC
May 2018

Lost in translation: returning germline genetic results in genome-scale cancer research.

Genome Med 2017 04 28;9(1):41. Epub 2017 Apr 28.

Cancer Research Program, Garvan Institute of Medical Research, Kinghorn Cancer Centre, Sydney, Australia.

Background: The return of research results (RoR) remains a complex and well-debated issue. Despite the debate, actual data related to the experience of giving individual results back, and the impact these results may have on clinical care and health outcomes, is sorely lacking. Through the work of the Australian Pancreatic Cancer Genome Initiative (APGI) we: (1) delineate the pathway back to the patient where actionable research data were identified; and (2) report the clinical utilisation of individual results returned. Using this experience, we discuss barriers and opportunities associated with a comprehensive process of RoR in large-scale genomic research that may be useful for others developing their own policies.

Methods: We performed whole-genome (n = 184) and exome (n = 208) sequencing of matched tumour-normal DNA pairs from 392 patients with sporadic pancreatic cancer (PC) as part of the APGI. We identified pathogenic germline mutations in candidate genes (n = 130) with established predisposition to PC or medium-high penetrance genes with well-defined cancer associated syndromes or phenotypes. Variants from candidate genes were annotated and classified according to international guidelines. Variants were considered actionable if clinical utility was established, with regard to prevention, diagnosis, prognostication and/or therapy.

Results: A total of 48,904 germline variants were identified, with 2356 unique variants undergoing annotation and in silico classification. Twenty cases were deemed actionable and were returned via previously described RoR framework, representing an actionable finding rate of 5.1%. Overall, 1.78% of our cohort experienced clinical benefit from RoR.

Conclusion: Returning research results within the context of large-scale genomics research is a labour-intensive, highly variable, complex operation. Results that warrant action are not infrequent, but the prevalence of those who experience a clinical difference as a result of returning individual results is currently low.
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http://dx.doi.org/10.1186/s13073-017-0430-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5408494PMC
April 2017

Hypermutation In Pancreatic Cancer.

Gastroenterology 2017 01 15;152(1):68-74.e2. Epub 2016 Nov 15.

QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia; Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland, Australia.

Pancreatic cancer is molecularly diverse, with few effective therapies. Increased mutation burden and defective DNA repair are associated with response to immune checkpoint inhibitors in several other cancer types. We interrogated 385 pancreatic cancer genomes to define hypermutation and its causes. Mutational signatures inferring defects in DNA repair were enriched in those with the highest mutation burdens. Mismatch repair deficiency was identified in 1% of tumors harboring different mechanisms of somatic inactivation of MLH1 and MSH2. Defining mutation load in individual pancreatic cancers and the optimal assay for patient selection may inform clinical trial design for immunotherapy in pancreatic cancer.
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http://dx.doi.org/10.1053/j.gastro.2016.09.060DOI Listing
January 2017

Case report of whole genome sequencing in the XY female: identification of a novel SRY mutation and revision of a misdiagnosis of androgen insensitivity syndrome.

BMC Endocr Disord 2016 Nov 8;16(1):58. Epub 2016 Nov 8.

Endocrine and Metabolic Unit, Royal Adelaide Hospital, Adelaide, SA, Australia.

Background: The 46,XY female is characterised by a male karyotype and female phenotype arising due to any interruption in the sexual development pathways in utero. The cause is usually genetic and various genes are implicated.

Case Presentation: Herein we describe a 46,XY woman who was first diagnosed with androgen insensitivity syndrome (testicular feminisation) at 18 years; however, this was later questioned due to the presence of intact Müllerian structures. The clinical phenotype suggested several susceptibility genes including SRY, DHH, NR5A1, NR0B1, AR, AMH, and AMHR2. To study candidate genes simultaneously, we performed whole genome sequencing. This revealed a novel and likely pathogenic missense variant (p.Arg130Pro, c.389G>C) in SRY, one of the major genes implicated in complete gonadal dysgenesis, hence securing this condition over androgen insensitivity syndrome as the cause of the patient's disorder of sexual development.

Conclusion: This case highlights the emerging clinical utility of whole genome sequencing as a tool in differentiating disorders of sexual development.
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http://dx.doi.org/10.1186/s12902-016-0141-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5100246PMC
November 2016

Genomic analyses identify molecular subtypes of pancreatic cancer.

Nature 2016 Mar 24;531(7592):47-52. Epub 2016 Feb 24.

Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, The University of Queensland, St Lucia, Brisbane, Queensland 4072, Australia.

Integrated genomic analysis of 456 pancreatic ductal adenocarcinomas identified 32 recurrently mutated genes that aggregate into 10 pathways: KRAS, TGF-β, WNT, NOTCH, ROBO/SLIT signalling, G1/S transition, SWI-SNF, chromatin modification, DNA repair and RNA processing. Expression analysis defined 4 subtypes: (1) squamous; (2) pancreatic progenitor; (3) immunogenic; and (4) aberrantly differentiated endocrine exocrine (ADEX) that correlate with histopathological characteristics. Squamous tumours are enriched for TP53 and KDM6A mutations, upregulation of the TP63∆N transcriptional network, hypermethylation of pancreatic endodermal cell-fate determining genes and have a poor prognosis. Pancreatic progenitor tumours preferentially express genes involved in early pancreatic development (FOXA2/3, PDX1 and MNX1). ADEX tumours displayed upregulation of genes that regulate networks involved in KRAS activation, exocrine (NR5A2 and RBPJL), and endocrine differentiation (NEUROD1 and NKX2-2). Immunogenic tumours contained upregulated immune networks including pathways involved in acquired immune suppression. These data infer differences in the molecular evolution of pancreatic cancer subtypes and identify opportunities for therapeutic development.
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http://dx.doi.org/10.1038/nature16965DOI Listing
March 2016

Presentation of m.3243A>G (MT-TL1; tRNALeu) variant with focal neurology in infancy.

Am J Med Genet A 2015 Nov 20;167A(11):2697-701. Epub 2015 Aug 20.

Department of Paediatrics, School of Paediatrics and Reproductive Health, University of Adelaide, North Adelaide, Australia.

The Mitochondrial tRNALeu (MT-TL1) mutation, m.3243A>G constitutes the commonest identified mitochondrial genome mutation. Characteristically, giving rise to MELAS (mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes), a phenotypic spectrum associated with this genetic variant is now apparent. We report on the first patient with infantile hemiparesis, without comorbid encephalopathy, attributed to this variant. This further expands the recognized disease spectrum and highlights the need to consider mitochondrial genomic mutations in cases of cryptogenic focal neurological deficit in infancy. The potential for genetic disease modifiers is additionally discussed.
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http://dx.doi.org/10.1002/ajmg.a.37161DOI Listing
November 2015

Integrated genomic and transcriptomic analysis of human brain metastases identifies alterations of potential clinical significance.

J Pathol 2015 Nov 19;237(3):363-78. Epub 2015 Aug 19.

QIMR Berghofer Medical Research Institute, Herston, Queensland, Australia.

Treatment options for patients with brain metastases (BMs) have limited efficacy and the mortality rate is virtually 100%. Targeted therapy is critically under-utilized, and our understanding of mechanisms underpinning metastatic outgrowth in the brain is limited. To address these deficiencies, we investigated the genomic and transcriptomic landscapes of 36 BMs from breast, lung, melanoma and oesophageal cancers, using DNA copy-number analysis and exome- and RNA-sequencing. The key findings were as follows. (a) Identification of novel candidates with possible roles in BM development, including the significantly mutated genes DSC2, ST7, PIK3R1 and SMC5, and the DNA repair, ERBB-HER signalling, axon guidance and protein kinase-A signalling pathways. (b) Mutational signature analysis was applied to successfully identify the primary cancer type for two BMs with unknown origins. (c) Actionable genomic alterations were identified in 31/36 BMs (86%); in one case we retrospectively identified ERBB2 amplification representing apparent HER2 status conversion, then confirmed progressive enrichment for HER2-positivity across four consecutive metastatic deposits by IHC and SISH, resulting in the deployment of HER2-targeted therapy for the patient. (d) In the ERBB/HER pathway, ERBB2 expression correlated with ERBB3 (r(2)  = 0.496; p < 0.0001) and HER3 and HER4 were frequently activated in an independent cohort of 167 archival BM from seven primary cancer types: 57.6% and 52.6% of cases were phospho-HER3(Y1222) or phospho-HER4(Y1162) membrane-positive, respectively. The HER3 ligands NRG1/2 were barely detectable by RNAseq, with NRG1 (8p12) genomic loss in 63.6% breast cancer-BMs, suggesting a microenvironmental source of ligand. In summary, this is the first study to characterize the genomic landscapes of BM. The data revealed novel candidates, potential clinical applications for genomic profiling of resectable BMs, and highlighted the possibility of therapeutically targeting HER3, which is broadly over-expressed and activated in BMs, independent of primary site and systemic therapy.
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http://dx.doi.org/10.1002/path.4583DOI Listing
November 2015

Whole-genome characterization of chemoresistant ovarian cancer.

Nature 2015 May;521(7553):489-94

Victorian Institute of Forensic Medicine, Southbank, Victoria 3006, Australia.

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.
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http://dx.doi.org/10.1038/nature14410DOI Listing
May 2015

Whole genomes redefine the mutational landscape of pancreatic cancer.

Nature 2015 Feb;518(7540):495-501

Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, The University of Queensland, St Lucia, Brisbane, Queensland 4072, Australia.

Pancreatic cancer remains one of the most lethal of malignancies and a major health burden. We performed whole-genome sequencing and copy number variation (CNV) analysis of 100 pancreatic ductal adenocarcinomas (PDACs). Chromosomal rearrangements leading to gene disruption were prevalent, affecting genes known to be important in pancreatic cancer (TP53, SMAD4, CDKN2A, ARID1A and ROBO2) and new candidate drivers of pancreatic carcinogenesis (KDM6A and PREX2). Patterns of structural variation (variation in chromosomal structure) classified PDACs into 4 subtypes with potential clinical utility: the subtypes were termed stable, locally rearranged, scattered and unstable. A significant proportion harboured focal amplifications, many of which contained druggable oncogenes (ERBB2, MET, FGFR1, CDK6, PIK3R3 and PIK3CA), but at low individual patient prevalence. Genomic instability co-segregated with inactivation of DNA maintenance genes (BRCA1, BRCA2 or PALB2) and a mutational signature of DNA damage repair deficiency. Of 8 patients who received platinum therapy, 4 of 5 individuals with these measures of defective DNA maintenance responded.
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http://dx.doi.org/10.1038/nature14169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4523082PMC
February 2015

Returning individual research results for genome sequences of pancreatic cancer.

Genome Med 2014 29;6(5):42. Epub 2014 May 29.

St John of God Subiaco, Perth, WA, 6008, Australia ; School of Surgery, The University of Western Australia, Perth, WA, 6009, Australia.

Background: Disclosure of individual results to participants in genomic research is a complex and contentious issue. There are many existing commentaries and opinion pieces on the topic, but little empirical data concerning actual cases describing how individual results have been returned. Thus, the real life risks and benefits of disclosing individual research results to participants are rarely if ever presented as part of this debate.

Methods: The Australian Pancreatic Cancer Genome Initiative (APGI) is an Australian contribution to the International Cancer Genome Consortium (ICGC), that involves prospective sequencing of tumor and normal genomes of study participants with pancreatic cancer in Australia. We present three examples that illustrate different facets of how research results may arise, and how they may be returned to individuals within an ethically defensible and clinically practical framework. This framework includes the necessary elements identified by others including consent, determination of the significance of results and which to return, delineation of the responsibility for communication and the clinical pathway for managing the consequences of returning results.

Results: Of 285 recruited patients, we returned results to a total of 25 with no adverse events to date. These included four that were classified as medically actionable, nine as clinically significant and eight that were returned at the request of the treating clinician. Case studies presented depict instances where research results impacted on cancer susceptibility, current treatment and diagnosis, and illustrate key practical challenges of developing an effective framework.

Conclusions: We suggest that return of individual results is both feasible and ethically defensible but only within the context of a robust framework that involves a close relationship between researchers and clinicians.
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http://dx.doi.org/10.1186/gm558DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4067993PMC
June 2014

Integrating massively parallel sequencing into diagnostic workflows and managing the annotation and clinical interpretation challenge.

Hum Mutat 2014 Apr 6;35(4):413-23. Epub 2014 Mar 6.

Genetic and Molecular Pathology, SA Pathology, Women's and Children's Hospital, North Adelaide, South Australia, 5006, Australia; School of Molecular and Biomedical Science, University of Adelaide, Adelaide, South Australia, 5000, Australia.

Massively parallel sequencing has become a powerful tool for the clinical management of patients with applications in diagnosis, guidance of treatment, prediction of drug response, and carrier screening. A considerable challenge for the clinical implementation of these technologies is the management of the vast amount of sequence data generated, in particular the annotation and clinical interpretation of genomic variants. Here, we describe annotation steps that can be automated and common strategies employed for variant prioritization. The definition of best practice standards for variant annotation and prioritization is still ongoing; at present, there is limited consensus regarding an optimal clinical sequencing pipeline. We provide considerations to help define these. For the first time, clinical genetics and genomics is not limited by our ability to sequence, but our ability to clinically interpret and use genomic information in health management. We argue that the development of standardized variant annotation and interpretation approaches and software tools implementing these warrants further support. As we gain a better understanding of the significance of genomic variation through research, patients will be able to benefit from the full scope that these technologies offer.
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http://dx.doi.org/10.1002/humu.22525DOI Listing
April 2014

Genome-wide DNA methylation patterns in pancreatic ductal adenocarcinoma reveal epigenetic deregulation of SLIT-ROBO, ITGA2 and MET signaling.

Int J Cancer 2014 Sep 9;135(5):1110-8. Epub 2014 May 9.

Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, The University of Queensland, St Lucia, Brisbane, QLD, Australia.

The importance of epigenetic modifications such as DNA methylation in tumorigenesis is increasingly being appreciated. To define the genome-wide pattern of DNA methylation in pancreatic ductal adenocarcinomas (PDAC), we captured the methylation profiles of 167 untreated resected PDACs and compared them to a panel of 29 adjacent nontransformed pancreata using high-density arrays. A total of 11,634 CpG sites associated with 3,522 genes were significantly differentially methylated (DM) in PDAC and were capable of segregating PDAC from non-malignant pancreas, regardless of tumor cellularity. As expected, PDAC hypermethylation was most prevalent in the 5' region of genes (including the proximal promoter, 5'UTR and CpG islands). Approximately 33% DM genes showed significant inverse correlation with mRNA expression levels. Pathway analysis revealed an enrichment of aberrantly methylated genes involved in key molecular mechanisms important to PDAC: TGF-β, WNT, integrin signaling, cell adhesion, stellate cell activation and axon guidance. Given the recent discovery that SLIT-ROBO mutations play a clinically important role in PDAC, the role of epigenetic perturbation of axon guidance was pursued in more detail. Bisulfite amplicon deep sequencing and qRT-PCR expression analyses confirmed recurrent perturbation of axon guidance pathway genes SLIT2, SLIT3, ROBO1, ROBO3, ITGA2 and MET and suggests epigenetic suppression of SLIT-ROBO signaling and up-regulation of MET and ITGA2 expression. Hypomethylation of MET and ITGA2 correlated with high gene expression, which was associated with poor survival. These data suggest that aberrant methylation plays an important role in pancreatic carcinogenesis affecting core signaling pathways with potential implications for the disease pathophysiology and therapy.
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http://dx.doi.org/10.1002/ijc.28765DOI Listing
September 2014

Somatic point mutation calling in low cellularity tumors.

PLoS One 2013 8;8(11):e74380. Epub 2013 Nov 8.

Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia.

Somatic mutation calling from next-generation sequencing data remains a challenge due to the difficulties of distinguishing true somatic events from artifacts arising from PCR, sequencing errors or mis-mapping. Tumor cellularity or purity, sub-clonality and copy number changes also confound the identification of true somatic events against a background of germline variants. We have developed a heuristic strategy and software (http://www.qcmg.org/bioinformatics/qsnp/) for somatic mutation calling in samples with low tumor content and we show the superior sensitivity and precision of our approach using a previously sequenced cell line, a series of tumor/normal admixtures, and 3,253 putative somatic SNVs verified on an orthogonal platform.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0074380PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3826759PMC
March 2015

Clinical and molecular characterization of HER2 amplified-pancreatic cancer.

Genome Med 2013 31;5(8):78. Epub 2013 Aug 31.

Kinghorn Cancer Centre and Garvan Institute of Medical Research, Darlinghurst, Sydney, Australia ; Wolfson Wohl Cancer Research Centre, Institute of Cancer Sciences, University of Glasgow, Glasgow, UK ; West of Scotland Pancreatic Unit, Glasgow Royal Infirmary, Glasgow, UK.

Background: Pancreatic cancer is one of the most lethal and molecularly diverse malignancies. Repurposing of therapeutics that target specific molecular mechanisms in different disease types offers potential for rapid improvements in outcome. Although HER2 amplification occurs in pancreatic cancer, it is inadequately characterized to exploit the potential of anti-HER2 therapies.

Methods: HER2 amplification was detected and further analyzed using multiple genomic sequencing approaches. Standardized reference laboratory assays defined HER2 amplification in a large cohort of patients (n = 469) with pancreatic ductal adenocarcinoma (PDAC).

Results: An amplified inversion event (1 MB) was identified at the HER2 locus in a patient with PDAC. Using standardized laboratory assays, we established diagnostic criteria for HER2 amplification in PDAC, and observed a prevalence of 2%. Clinically, HER2- amplified PDAC was characterized by a lack of liver metastases, and a preponderance of lung and brain metastases. Excluding breast and gastric cancer, the incidence of HER2-amplified cancers in the USA is >22,000 per annum.

Conclusions: HER2 amplification occurs in 2% of PDAC, and has distinct features with implications for clinical practice. The molecular heterogeneity of PDAC implies that even an incidence of 2% represents an attractive target for anti-HER2 therapies, as options for PDAC are limited. Recruiting patients based on HER2 amplification, rather than organ of origin, could make trials of anti-HER2 therapies feasible in less common cancer types.
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http://dx.doi.org/10.1186/gm482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3978667PMC
May 2014

Computational approaches to identify functional genetic variants in cancer genomes.

Nat Methods 2013 Aug;10(8):723-9

Research Unit on Biomedical Informatics, University Pompeu Fabra, Barcelona, Spain.

The International Cancer Genome Consortium (ICGC) aims to catalog genomic abnormalities in tumors from 50 different cancer types. Genome sequencing reveals hundreds to thousands of somatic mutations in each tumor but only a minority of these drive tumor progression. We present the result of discussions within the ICGC on how to address the challenge of identifying mutations that contribute to oncogenesis, tumor maintenance or response to therapy, and recommend computational techniques to annotate somatic variants and predict their impact on cancer phenotype.
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http://dx.doi.org/10.1038/nmeth.2562DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3919555PMC
August 2013

Cerebellar output in zebrafish: an analysis of spatial patterns and topography in eurydendroid cell projections.

Front Neural Circuits 2013 1;7:53. Epub 2013 Apr 1.

School of Biomedical Sciences, The University of Queensland Brisbane, QLD, Australia.

The cerebellum is a brain region responsible for motor coordination and for refining motor programs. While a great deal is known about the structure and connectivity of the mammalian cerebellum, fundamental questions regarding its function in behavior remain unanswered. Recently, the zebrafish has emerged as a useful model organism for cerebellar studies, owing in part to the similarity in cerebellar circuits between zebrafish and mammals. While the cell types composing their cerebellar cortical circuits are generally conserved with mammals, zebrafish lack deep cerebellar nuclei, and instead a majority of cerebellar output comes from a single type of neuron: the eurydendroid cell. To describe spatial patterns of cerebellar output in zebrafish, we have used genetic techniques to label and trace eurydendroid cells individually and en masse. We have found that cerebellar output targets the thalamus and optic tectum, and have confirmed the presence of pre-synaptic terminals from eurydendroid cells in these structures using a synaptically targeted GFP. By observing individual eurydendroid cells, we have shown that different medial-lateral regions of the cerebellum have eurydendroid cells projecting to different targets. Finally, we found topographic organization in the connectivity between the cerebellum and the optic tectum, where more medial eurydendroid cells project to the rostral tectum while lateral cells project to the caudal tectum. These findings indicate that there is spatial logic underpinning cerebellar output in zebrafish with likely implications for cerebellar function.
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http://dx.doi.org/10.3389/fncir.2013.00053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3612595PMC
May 2014

Complex evolutionary relationships among four classes of modular RNA-binding splicing regulators in eukaryotes: the hnRNP, SR, ELAV-like and CELF proteins.

J Mol Evol 2012 Dec 24;75(5-6):214-28. Epub 2012 Nov 24.

School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, QLD, Australia.

Alternative RNA splicing in multicellular organisms is regulated by a large group of proteins of mainly unknown origin. To predict the functions of these proteins, classification of their domains at the sequence and structural level is necessary. We have focused on four groups of splicing regulators, the heterogeneous nuclear ribonucleoprotein (hnRNP), serine-arginine (SR), embryonic lethal, abnormal vision (ELAV)-like, and CUG-BP and ETR-like factor (CELF) proteins, that show increasing diversity among metazoa. Sequence and phylogenetic analyses were used to obtain a broader understanding of their evolutionary relationships. Surprisingly, when we characterised sequence similarities across full-length sequences and conserved domains of ten metazoan species, we found some hnRNPs were more closely related to SR, ELAV-like and CELF proteins than to other hnRNPs. Phylogenetic analyses and the distribution of the RRM domains suggest that these proteins diversified before the last common ancestor of the metazoans studied here through domain acquisition and duplication to create genes of mixed evolutionary origin. We propose that these proteins were derived independently rather than through the expansion of a single protein family. Our results highlight inconsistencies in the current classification system for these regulators, which does not adequately reflect their evolutionary relationships, and suggests that a domain-based classification scheme may have more utility.
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http://dx.doi.org/10.1007/s00239-012-9533-0DOI Listing
December 2012

Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes.

Nature 2012 Nov 24;491(7424):399-405. Epub 2012 Oct 24.

The Kinghorn Cancer Centre, 370 Victoria Street, Darlinghurst, Sydney, New South Wales 2010, Australia.

Pancreatic cancer is a highly lethal malignancy with few effective therapies. We performed exome sequencing and copy number analysis to define genomic aberrations in a prospectively accrued clinical cohort (n = 142) of early (stage I and II) sporadic pancreatic ductal adenocarcinoma. Detailed analysis of 99 informative tumours identified substantial heterogeneity with 2,016 non-silent mutations and 1,628 copy-number variations. We define 16 significantly mutated genes, reaffirming known mutations (KRAS, TP53, CDKN2A, SMAD4, MLL3, TGFBR2, ARID1A and SF3B1), and uncover novel mutated genes including additional genes involved in chromatin modification (EPC1 and ARID2), DNA damage repair (ATM) and other mechanisms (ZIM2, MAP2K4, NALCN, SLC16A4 and MAGEA6). Integrative analysis with in vitro functional data and animal models provided supportive evidence for potential roles for these genetic aberrations in carcinogenesis. Pathway-based analysis of recurrently mutated genes recapitulated clustering in core signalling pathways in pancreatic ductal adenocarcinoma, and identified new mutated genes in each pathway. We also identified frequent and diverse somatic aberrations in genes described traditionally as embryonic regulators of axon guidance, particularly SLIT/ROBO signalling, which was also evident in murine Sleeping Beauty transposon-mediated somatic mutagenesis models of pancreatic cancer, providing further supportive evidence for the potential involvement of axon guidance genes in pancreatic carcinogenesis.
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http://dx.doi.org/10.1038/nature11547DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3530898PMC
November 2012

qpure: A tool to estimate tumor cellularity from genome-wide single-nucleotide polymorphism profiles.

PLoS One 2012 25;7(9):e45835. Epub 2012 Sep 25.

Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, Queensland, Australia.

Tumour cellularity, the relative proportion of tumour and normal cells in a sample, affects the sensitivity of mutation detection, copy number analysis, cancer gene expression and methylation profiling. Tumour cellularity is traditionally estimated by pathological review of sectioned specimens; however this method is both subjective and prone to error due to heterogeneity within lesions and cellularity differences between the sample viewed during pathological review and tissue used for research purposes. In this paper we describe a statistical model to estimate tumour cellularity from SNP array profiles of paired tumour and normal samples using shifts in SNP allele frequency at regions of loss of heterozygosity (LOH) in the tumour. We also provide qpure, a software implementation of the method. Our experiments showed that there is a medium correlation 0.42 ([Formula: see text]-value=0.0001) between tumor cellularity estimated by qpure and pathology review. Interestingly there is a high correlation 0.87 ([Formula: see text]-value [Formula: see text] 2.2e-16) between cellularity estimates by qpure and deep Ion Torrent sequencing of known somatic KRAS mutations; and a weaker correlation 0.32 ([Formula: see text]-value=0.004) between IonTorrent sequencing and pathology review. This suggests that qpure may be a more accurate predictor of tumour cellularity than pathology review. qpure can be downloaded from https://sourceforge.net/projects/qpure/.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0045835PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3457972PMC
May 2013

The mammalian PYHIN gene family: phylogeny, evolution and expression.

BMC Evol Biol 2012 Aug 7;12:140. Epub 2012 Aug 7.

The University of Queensland, School of Chemistry and Molecular Biosciences, Brisbane, Qld 4072, Australia.

Background: Proteins of the mammalian PYHIN (IFI200/HIN-200) family are involved in defence against infection through recognition of foreign DNA. The family member absent in melanoma 2 (AIM2) binds cytosolic DNA via its HIN domain and initiates inflammasome formation via its pyrin domain. AIM2 lies within a cluster of related genes, many of which are uncharacterised in mouse. To better understand the evolution, orthology and function of these genes, we have documented the range of PYHIN genes present in representative mammalian species, and undertaken phylogenetic and expression analyses.

Results: No PYHIN genes are evident in non-mammals or monotremes, with a single member found in each of three marsupial genomes. Placental mammals show variable family expansions, from one gene in cow to four in human and 14 in mouse. A single HIN domain appears to have evolved in the common ancestor of marsupials and placental mammals, and duplicated to give rise to three distinct forms (HIN-A, -B and -C) in the placental mammal ancestor. Phylogenetic analyses showed that AIM2 HIN-C and pyrin domains clearly diverge from the rest of the family, and it is the only PYHIN protein with orthology across many species. Interestingly, although AIM2 is important in defence against some bacteria and viruses in mice, AIM2 is a pseudogene in cow, sheep, llama, dolphin, dog and elephant. The other 13 mouse genes have arisen by duplication and rearrangement within the lineage, which has allowed some diversification in expression patterns.

Conclusions: The role of AIM2 in forming the inflammasome is relatively well understood, but molecular interactions of other PYHIN proteins involved in defence against foreign DNA remain to be defined. The non-AIM2 PYHIN protein sequences are very distinct from AIM2, suggesting they vary in effector mechanism in response to foreign DNA, and may bind different DNA structures. The PYHIN family has highly varied gene composition between mammalian species due to lineage-specific duplication and loss, which probably indicates different adaptations for fighting infectious disease. Non-genomic DNA can indicate infection, or a mutagenic threat. We hypothesise that defence of the genome against endogenous retroelements has been an additional evolutionary driver for PYHIN proteins.
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http://dx.doi.org/10.1186/1471-2148-12-140DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3458909PMC
August 2012

Sleeping Beauty mutagenesis reveals cooperating mutations and pathways in pancreatic adenocarcinoma.

Proc Natl Acad Sci U S A 2012 Apr 15;109(16):5934-41. Epub 2012 Mar 15.

Division of Genetics and Genomics, Institute of Molecular and Cell Biology, Singapore 138673.

Pancreatic cancer is one of the most deadly cancers affecting the Western world. Because the disease is highly metastatic and difficult to diagnosis until late stages, the 5-y survival rate is around 5%. The identification of molecular cancer drivers is critical for furthering our understanding of the disease and development of improved diagnostic tools and therapeutics. We have conducted a mutagenic screen using Sleeping Beauty (SB) in mice to identify new candidate cancer genes in pancreatic cancer. By combining SB with an oncogenic Kras allele, we observed highly metastatic pancreatic adenocarcinomas. Using two independent statistical methods to identify loci commonly mutated by SB in these tumors, we identified 681 loci that comprise 543 candidate cancer genes (CCGs); 75 of these CCGs, including Mll3 and Ptk2, have known mutations in human pancreatic cancer. We identified point mutations in human pancreatic patient samples for another 11 CCGs, including Acvr2a and Map2k4. Importantly, 10% of the CCGs are involved in chromatin remodeling, including Arid4b, Kdm6a, and Nsd3, and all SB tumors have at least one mutated gene involved in this process; 20 CCGs, including Ctnnd1, Fbxo11, and Vgll4, are also significantly associated with poor patient survival. SB mutagenesis provides a rich resource of mutations in potential cancer drivers for cross-comparative analyses with ongoing sequencing efforts in human pancreatic adenocarcinoma.
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http://dx.doi.org/10.1073/pnas.1202490109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3341075PMC
April 2012

MicroRNAs and their isomiRs function cooperatively to target common biological pathways.

Genome Biol 2011 Dec 30;12(12):R126. Epub 2011 Dec 30.

Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, The University of Queensland, St Lucia, Queensland 4072, Australia.

Background: Variants of microRNAs (miRNAs), called isomiRs, are commonly reported in deep-sequencing studies; however, the functional significance of these variants remains controversial. Observational studies show that isomiR patterns are non-random, hinting that these molecules could be regulated and therefore functional, although no conclusive biological role has been demonstrated for these molecules.

Results: To assess the biological relevance of isomiRs, we have performed ultra-deep miRNA-seq on ten adult human tissues, and created an analysis pipeline called miRNA-MATE to align, annotate, and analyze miRNAs and their isomiRs. We find that isomiRs share sequence and expression characteristics with canonical miRNAs, and are generally strongly correlated with canonical miRNA expression. A large proportion of isomiRs potentially derive from AGO2 cleavage independent of Dicer. We isolated polyribosome-associated mRNA, captured the mRNA-bound miRNAs, and found that isomiRs and canonical miRNAs are equally associated with translational machinery. Finally, we transfected cells with biotinylated RNA duplexes encoding isomiRs or their canonical counterparts and directly assayed their mRNA targets. These studies allow us to experimentally determine genome-wide mRNA targets, and these experiments showed substantial overlap in functional mRNA networks suppressed by both canonical miRNAs and their isomiRs.

Conclusions: Together, these results find isomiRs to be biologically relevant and functionally cooperative partners of canonical miRNAs that act coordinately to target pathways of functionally related genes. This work exposes the complexity of the miRNA-transcriptome, and helps explain a major miRNA paradox: how specific regulation of biological processes can occur when the specificity of miRNA targeting is mediated by only 6 to 11 nucleotides.
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http://dx.doi.org/10.1186/gb-2011-12-12-r126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3334621PMC
December 2011

PINA v2.0: mining interactome modules.

Nucleic Acids Res 2012 Jan 8;40(Database issue):D862-5. Epub 2011 Nov 8.

Cancer Research Program, Peter Wills Bioinformatics Centre, Garvan Institute of Medical Research, Darlinghurst, Sydney, NSW 2010, Australia.

The Protein Interaction Network Analysis (PINA) platform is a comprehensive web resource, which includes a database of unified protein-protein interaction data integrated from six manually curated public databases, and a set of built-in tools for network construction, filtering, analysis and visualization. The second version of PINA enhances its utility for studies of protein interactions at a network level, by including multiple collections of interaction modules identified by different clustering approaches from the whole network of protein interactions ('interactome') for six model organisms. All identified modules are fully annotated by enriched Gene Ontology terms, KEGG pathways, Pfam domains and the chemical and genetic perturbations collection from MSigDB. Moreover, a new tool is provided for module enrichment analysis in addition to simple query function. The interactome data are also available on the web site for further bioinformatics analysis. PINA is freely accessible at http://cbg.garvan.unsw.edu.au/pina/.
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http://dx.doi.org/10.1093/nar/gkr967DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3244997PMC
January 2012

Deep-transcriptome and ribonome sequencing redefines the molecular networks of pluripotency and the extracellular space in human embryonic stem cells.

Genome Res 2011 Dec 31;21(12):2014-25. Epub 2011 Oct 31.

Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, The University of Queensland, Queensland 4072, Australia.

Recent RNA-sequencing studies have shown remarkable complexity in the mammalian transcriptome. The ultimate impact of this complexity on the predicted proteomic output is less well defined. We have undertaken strand-specific RNA sequencing of multiple cellular RNA fractions (>20 Gb) to uncover the transcriptional complexity of human embryonic stem cells (hESCs). We have shown that human embryonic stem (ES) cells display a high degree of transcriptional diversity, with more than half of active genes generating RNAs that differ from conventional gene models. We found evidence that more than 1000 genes express long 5' and/or extended 3'UTRs, which was confirmed by "virtual Northern" analysis. Exhaustive sequencing of the membrane-polysome and cytosolic/untranslated fractions of hESCs was used to identify RNAs encoding peptides destined for secretion and the extracellular space and to demonstrate preferential selection of transcription complexity for translation in vitro. The impact of this newly defined complexity on known gene-centric network models such as the Plurinet and the cell surface signaling machinery in human ES cells revealed a significant expansion of known transcript isoforms at play, many predicting possible alternative functions based on sequence alterations within key functional domains.
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http://dx.doi.org/10.1101/gr.119321.110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3227092PMC
December 2011

Mineralocorticoid receptors: evolutionary and pathophysiological considerations.

Endocrinology 2011 May 22;152(5):1883-90. Epub 2011 Feb 22.

Institute for Molecular Bioscience and Australian Research Council Centre of Excellence in Bioinformatic, University of Queensland, Brisbane, Queensland 4072, Australia.

Mineralocorticoid receptors (MR), glucocorticoid receptors (GR), progesterone receptors (PR), and androgen receptors (AR) comprise a closely related subfamily within the human 49-member nuclear receptor family. These receptors and their cognate ligands play major roles in homeostasis, reproduction, growth, and development, despite which their evolution and diversification remains incompletely understood. Several conflicting models have been advanced for the evolution of this subfamily. We have thus undertaken Bayesian and maximum likelihood phylogenetic analyses of this subfamily. The Bayesian consensus and maximum likelihood trees support a basal position for MR, with the PR and AR forming a sister clade. We next performed analyses using topological constraints to directly contrast the likelihood of seven phylogenetic models. In these analyses, three models have similar support: one proposes two sister clades (MR and GR, PR and AR); the other two propose a different subfamily member (MR or GR) to be the first to have diverged. Ancestral state reconstructions at sites critical for physiological function show that the S810L mutation in the MR, which results in the MR being similar to estrogen receptors and the more distantly related retinoic acid receptor-α is likely to reflect the ancestral receptor sequence before the divergence of this subfamily and provides further support for MR having been the first of the subfamily to diverge. Finally, we drew on pathophysiological comparisons to help to distinguish the different models. On the basis of our phylogenetic analyses and pathophysiological considerations, we propose that the MR was the first to diverge from the ancestral gene lineage from which this subfamily derived.
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http://dx.doi.org/10.1210/en.2010-1444DOI Listing
May 2011

Sequencing transcriptomes in toto.

Integr Biol (Camb) 2011 May 4;3(5):522-8. Epub 2011 Feb 4.

Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, Brisbane, Australia.

The development of next-generation sequencing technologies has enabled the transcriptome to be measured and characterized at a level which was previously unattainable. Shot gun sequencing of RNAs, or RNA-Seq as it is known, is providing the means to simultaneously survey locus activity, transcript-specific expression, sequence content of transcripts and transcriptome discovery. This article discusses the current state of RNA-Seq, its potential for redefining transcriptomics and some of the challenges associated with this revolutionary technology.
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http://dx.doi.org/10.1039/c0ib00062kDOI Listing
May 2011

Functional implications of the emergence of alternative splicing in hnRNP A/B transcripts.

RNA 2010 Sep 22;16(9):1760-8. Epub 2010 Jul 22.

School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, QLD 4072, Australia.

The heterogeneous nuclear ribonucleoproteins (hnRNPs) A/B are a family of RNA-binding proteins that participate in various aspects of nucleic acid metabolism, including mRNA trafficking, telomere maintenance, and splicing. They are both regulators and targets of alternative splicing, and the patterns of alternative splicing of their transcripts have diverged between paralogs and between orthologs in different species. Surprisingly, the extent of this splicing variation and its implications for post-transcriptional regulation have remained largely unexplored. Here, we conducted a detailed analysis of hnRNP A/B sequences and expression patterns across six vertebrates. Alternative exons emerged via the introduction of new splice sites, changes in the strengths of existing splice sites, and the accumulation of auxiliary splicing regulatory motifs. Observed isoform expression patterns could be attributed to the frequency and strength of cis-elements. We found a trend toward increased splicing variation in mammals and identified novel alternatively spliced isoforms in human and chicken. Pulldown and translational assays demonstrated that the inclusion of alternative exons altered the affinity of hnRNP A/B proteins for their cognate nucleic acids and modified protein expression levels. As the hnRNPs A/B regulate several key steps in mRNA processing, the involvement of diverse hnRNP isoforms in multiple cellular contexts and species implies concomitant differences in the transcriptional output of these systems. We conclude that the emergence of alternative splicing in the hnRNPs A/B has contributed to the diversification of their roles in the regulation of alternative splicing and has thus added an unexpected layer of regulatory complexity to transcription in vertebrates.
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http://dx.doi.org/10.1261/rna.2142810DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2924535PMC
September 2010

A visual framework for sequence analysis using n-grams and spectral rearrangement.

Bioinformatics 2010 Mar 3;26(6):737-44. Epub 2010 Feb 3.

Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia.

Motivation: Protein sequences are often composed of regions that have distinct evolutionary histories as a consequence of domain shuffling, recombination or gene conversion. New approaches are required to discover, visualize and analyze these sequence regions and thus enable a better understanding of protein evolution.

Results: Here, we have developed an alignment-free and visual approach to analyze sequence relationships. We use the number of shared n-grams between sequences as a measure of sequence similarity and rearrange the resulting affinity matrix applying a spectral technique. Heat maps of the affinity matrix are employed to identify and visualize clusters of related sequences or outliers, while n-gram-based dot plots and conservation profiles allow detailed analysis of similarities among selected sequences. Using this approach, we have identified signatures of domain shuffling in an otherwise poorly characterized family, and homology clusters in another. We conclude that this approach may be generally useful as a framework to analyze related, but highly divergent protein sequences. It is particularly useful as a fast method to study sequence relationships prior to much more time-consuming multiple sequence alignment and phylogenetic analysis.

Availability: A software implementation (MOSAIC) of the framework described here can be downloaded from http://bioinformatics.org.au/mosaic/

Contact: [email protected]

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btq042DOI Listing
March 2010
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