- Email: [email protected]
Defining the role of rare, non-coding DNA variation in disease
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PATHOLOGY 2014 ABSTRACT SUPPLEMENT
identify the genetic cause in 97 (65%) of these patients and in doing so identified 10 novel disease genes. This talk will focus on our ‘MitoExome’ targeted MPS strategy, which we have used to sequence 1000 genes encoding the mitochondrial proteome in 44 patients.1,2 In particular, I will focus on the strategies used for variant filtering and prioritisation, how the variants are then validated and what needs to be done to confirm pathogenicity. I will also discuss some cases where our approach was unable to detect the disease causing variants. References 1. Calvo SE, Compton AG, Hershman SG, et al. Molecular diagnosis of infantile mitochondrial disease with targeted next-generation sequencing. Sci Transl Med 2012; 4: 118ra10. 2. Tucker EJ, Hershman SG, Ko¨hrer C, et al. Mutations in MTFMT underlie a human disorder of formylation causing impaired mitochondrial translation. Cell Metabolism 2011; 14: 428–34.
INVESTIGATION OF CARDIAC DEATH – WHAT IS (TECHNICALLY) POSSIBLE? E. P. Kirk1,2,3 1Department of Medical Genetics, Sydney Children’s Hospital, 2South Eastern Area Laboratory Services, NSW Health Pathology, Randwick, and 3School of Women’s and Children’s Health, University of New South Wales, Randwick, NSW, Australia In searching for genetic causes of cardiac death, the key requirements are collection of the appropriate sample and selection of the appropriate test. It is essential to obtain a good source of DNA; occasionally, establishing a fibroblast culture may be possible and worthwhile. Once DNA is available, options for mutation screening are changing rapidly, as new sequencing technologies move into the diagnostic laboratory. Selecting a genetic test involves not just the history and post-mortem findings, but ideally should also rely on evaluation of the family, including family history and potentially investigation of family members. The results of testing may have important implications for family members. Options range from no genetic testing through sequencing a single gene, sequencing (increasingly large) panels of genes, and even sequencing essentially all of the known genes (exome sequencing). A negative result on a test done today is not necessarily the end of the story – the options available in 5 years (and our ability to interpret the results) are likely to be substantially different from the situation today. In addition to discussion of these issues, this talk will include a shopper’s guide to currently available tests, mosquitoes in amber and a 700,000 year old horse. ASSESSING THE CAUSALITY OF GENETIC VARIANTS IN CARDIAC DISEASE GENES Paul A. James Genetic Medicine, Royal Melbourne Hospital, Melbourne, Vic, Australia The genetics of inherited cardiac disorders is complex with many conditions demonstrating extensive genetic heterogeneity. Increasingly broad approaches are being taken to genetic testing for these conditions but the larger number of genes involved brings with it greater difficulty arriving at clinically certain interpretations of the
Pathology (2014), 46(S1)
data. This problem is amplified in the setting of sudden unexpected death (SUD) where, by definition, there are few clinical clues to provide guidance. Recent publications have highlighted the fallibility of some mutation calls in the past and raised concerns about errors being perpetuated through the literature. To overcome these issues rigorous approaches have been developed that aim to integrate data from a number of independent areas to improve the final interpretation and reporting of variants. In this talk I will look at the information generated by analysis of physicochemical properties (Grantham analysis), evolutionary conservation (e.g., SIFT, Polyphen, A_GVGD), segregation analysis and functional studies, using examples from sequencing data from more than 25,000 genes screened using an NGS gene panel available for clinical use, including sudden cardiac death. I will consider the strength and weaknesses of these approaches and illustrate how they can be combined to improve the quality of reporting. LONG NON-CODING RNAS IN DISEASE AND DEVELOPMENT Marcel E. Dinger1,2 1Garvan Institute of Medical Research, Darlinghurst, and 2St Vincent’s Clinical School, University of New South Wales, Kensington, NSW, Australia Approximately 98% of the human genome comprises non-coding DNA, the function of which is largely unknown. Intriguingly, more than 85% of single nucleotide polymorphisms identified as diseaseassociated by genome-wide association studies (GWAS) occur in non-coding regions. The relatively recent discovery of widespread transcription of potentially functional long non-coding RNAs (lncRNAs) led us to investigate whether or not GWAS hits in non-coding regions could be reconciled by the transcription of regulatory RNAs. As lncRNAs typically show highly developmental-stage- and tissuespecific expression, they cannot be easily detected by RNA-Seq, which requires exponentially greater depth to detect increasingly rare transcripts. To overcome this problem, we developed a technique termed RNA-Capture-Seq,1 which targets RNA sequencing to specific areas of the genome. We have used this approach to target 300 chromosomal regions identified by GWAS. Using RNA from diverse human tissues, we identify thousands of novel differentially expressed transcripts. Although functional investigation of these transcripts is still underway, these experiments bring an intriguing new perspective into our understanding of how information in the genome is encoded and have considerable potential to identify novel regulators, which may prove valuable as biomarkers and therapeutic targets, involved in disease and development. Reference
1. Mercer TR, Gerhardt DJ, Dinger ME, et al. Targeted RNA sequencing reveals the deep complexity of the human transcriptome. Nat Biotechnol 2011; 30: 99–104.
DEFINING THE ROLE OF RARE, NON-CODING DNA VARIATION IN DISEASE Stefan White Center for Genetic Diseases, Monash Institute of Medical Research, Monash University, Clayton, Vic, Australia
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
ABSTRACTS
The development of high-density microarrays and next-generation sequencing technologies has revealed the true extent of human genetic variation. Not only does every genome contain millions of single nucleotide variants, but there are also a large number of copy number variants (CNVs). This variation contributes to what makes each individual unique, but in some cases also plays a role in disease. Determining the effect of a coding change is relatively straightforward in many cases, however predicting the consequence of variants affecting noncoding DNA is more problematic. This presentation will describe a range of methods we have used for studying different types of genetic variation, and outline examples of rare CNVs and single nucleotide variants in non-coding DNA that are associated with specific conditions.
ENSURING CLINICAL VALIDITY Elaine R. Mardis Genome Institute, Washington University School of Medicine, St Louis, MO, USA Transitioning a technology like next-generation sequencing, with its attendant preparatory procedures, data volumes, and analytical overhead into a clinical environment presents new and unique challenges. The application of comprehensive assays in the diagnostic setting involves multiple disciplines and a sequencing pipeline that moves smoothly from sample intake through the completion of a molecular diagnostic ‘report’ with pathology sign-off. My lecture will focus on specific details of our current diagnostic approaches, how they are presented to the signing pathologist, and how we maintain the integrity and clinical validity of the variants we report.
S27
ENSURING CLINICAL VALIDITY – MODERNISING GENETIC TESTING SERVICES Karin S. Kassahn1, Hamish S. Scott1,2 and Janice M. Fletcher1,3 1Genetic and Molecular Pathology, SA Pathology, 2Centre for Cancer Biology, SA Pathology, and 3School of Paediatrics and Reproductive Health, Faculty of Health Sciences, The University of Adelaide, Adelaide, SA, Australia Genomic testing using gene panels and whole-exome sequence data are becoming increasingly attractive options to support the clinical management of patients. Whole-exome sequencing to diagnose rare genetic disorders in paediatrics offers a cost-effective and efficient first screen for cases where there is no obvious clinical diagnosis, thus fundamentally shifting medical service provision paradigms. In other areas of medicine, the implementation of targeted gene panels using next generation sequencing is associated with improved diagnosis rate and faster time to diagnosis. Successful integration of genomic testing into the patient management pathway requires updating of the traditional models of test provision. Important factors to be considered include: who should qualify for what genomic test, payment for testing, the appropriate referral mechanism, and how to effectively communicate the results and limitations of genomic tests to patients and clinicians. We discuss strategies and give examples from implementing gene panels and whole-exome sequencing in clinical diagnostics to support the management of patients in South Australia. The next few years are likely to be an exciting and challenging time in genomic test provision in Australia, as these new platforms establish themselves as part of routine medical care and different stakeholders (local, international, direct-to-consumer) define their share of the clinical sequencing market.
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
PATHOLOGY 2014 ABSTRACT SUPPLEMENT
identify the genetic cause in 97 (65%) of these patients and in doing so identified 10 novel disease genes. This talk will focus on our ‘MitoExome’ targeted MPS strategy, which we have used to sequence 1000 genes encoding the mitochondrial proteome in 44 patients.1,2 In particular, I will focus on the strategies used for variant filtering and prioritisation, how the variants are then validated and what needs to be done to confirm pathogenicity. I will also discuss some cases where our approach was unable to detect the disease causing variants. References 1. Calvo SE, Compton AG, Hershman SG, et al. Molecular diagnosis of infantile mitochondrial disease with targeted next-generation sequencing. Sci Transl Med 2012; 4: 118ra10. 2. Tucker EJ, Hershman SG, Ko¨hrer C, et al. Mutations in MTFMT underlie a human disorder of formylation causing impaired mitochondrial translation. Cell Metabolism 2011; 14: 428–34.
INVESTIGATION OF CARDIAC DEATH – WHAT IS (TECHNICALLY) POSSIBLE? E. P. Kirk1,2,3 1Department of Medical Genetics, Sydney Children’s Hospital, 2South Eastern Area Laboratory Services, NSW Health Pathology, Randwick, and 3School of Women’s and Children’s Health, University of New South Wales, Randwick, NSW, Australia In searching for genetic causes of cardiac death, the key requirements are collection of the appropriate sample and selection of the appropriate test. It is essential to obtain a good source of DNA; occasionally, establishing a fibroblast culture may be possible and worthwhile. Once DNA is available, options for mutation screening are changing rapidly, as new sequencing technologies move into the diagnostic laboratory. Selecting a genetic test involves not just the history and post-mortem findings, but ideally should also rely on evaluation of the family, including family history and potentially investigation of family members. The results of testing may have important implications for family members. Options range from no genetic testing through sequencing a single gene, sequencing (increasingly large) panels of genes, and even sequencing essentially all of the known genes (exome sequencing). A negative result on a test done today is not necessarily the end of the story – the options available in 5 years (and our ability to interpret the results) are likely to be substantially different from the situation today. In addition to discussion of these issues, this talk will include a shopper’s guide to currently available tests, mosquitoes in amber and a 700,000 year old horse. ASSESSING THE CAUSALITY OF GENETIC VARIANTS IN CARDIAC DISEASE GENES Paul A. James Genetic Medicine, Royal Melbourne Hospital, Melbourne, Vic, Australia The genetics of inherited cardiac disorders is complex with many conditions demonstrating extensive genetic heterogeneity. Increasingly broad approaches are being taken to genetic testing for these conditions but the larger number of genes involved brings with it greater difficulty arriving at clinically certain interpretations of the
Pathology (2014), 46(S1)
data. This problem is amplified in the setting of sudden unexpected death (SUD) where, by definition, there are few clinical clues to provide guidance. Recent publications have highlighted the fallibility of some mutation calls in the past and raised concerns about errors being perpetuated through the literature. To overcome these issues rigorous approaches have been developed that aim to integrate data from a number of independent areas to improve the final interpretation and reporting of variants. In this talk I will look at the information generated by analysis of physicochemical properties (Grantham analysis), evolutionary conservation (e.g., SIFT, Polyphen, A_GVGD), segregation analysis and functional studies, using examples from sequencing data from more than 25,000 genes screened using an NGS gene panel available for clinical use, including sudden cardiac death. I will consider the strength and weaknesses of these approaches and illustrate how they can be combined to improve the quality of reporting. LONG NON-CODING RNAS IN DISEASE AND DEVELOPMENT Marcel E. Dinger1,2 1Garvan Institute of Medical Research, Darlinghurst, and 2St Vincent’s Clinical School, University of New South Wales, Kensington, NSW, Australia Approximately 98% of the human genome comprises non-coding DNA, the function of which is largely unknown. Intriguingly, more than 85% of single nucleotide polymorphisms identified as diseaseassociated by genome-wide association studies (GWAS) occur in non-coding regions. The relatively recent discovery of widespread transcription of potentially functional long non-coding RNAs (lncRNAs) led us to investigate whether or not GWAS hits in non-coding regions could be reconciled by the transcription of regulatory RNAs. As lncRNAs typically show highly developmental-stage- and tissuespecific expression, they cannot be easily detected by RNA-Seq, which requires exponentially greater depth to detect increasingly rare transcripts. To overcome this problem, we developed a technique termed RNA-Capture-Seq,1 which targets RNA sequencing to specific areas of the genome. We have used this approach to target 300 chromosomal regions identified by GWAS. Using RNA from diverse human tissues, we identify thousands of novel differentially expressed transcripts. Although functional investigation of these transcripts is still underway, these experiments bring an intriguing new perspective into our understanding of how information in the genome is encoded and have considerable potential to identify novel regulators, which may prove valuable as biomarkers and therapeutic targets, involved in disease and development. Reference
1. Mercer TR, Gerhardt DJ, Dinger ME, et al. Targeted RNA sequencing reveals the deep complexity of the human transcriptome. Nat Biotechnol 2011; 30: 99–104.
DEFINING THE ROLE OF RARE, NON-CODING DNA VARIATION IN DISEASE Stefan White Center for Genetic Diseases, Monash Institute of Medical Research, Monash University, Clayton, Vic, Australia
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
ABSTRACTS
The development of high-density microarrays and next-generation sequencing technologies has revealed the true extent of human genetic variation. Not only does every genome contain millions of single nucleotide variants, but there are also a large number of copy number variants (CNVs). This variation contributes to what makes each individual unique, but in some cases also plays a role in disease. Determining the effect of a coding change is relatively straightforward in many cases, however predicting the consequence of variants affecting noncoding DNA is more problematic. This presentation will describe a range of methods we have used for studying different types of genetic variation, and outline examples of rare CNVs and single nucleotide variants in non-coding DNA that are associated with specific conditions.
ENSURING CLINICAL VALIDITY Elaine R. Mardis Genome Institute, Washington University School of Medicine, St Louis, MO, USA Transitioning a technology like next-generation sequencing, with its attendant preparatory procedures, data volumes, and analytical overhead into a clinical environment presents new and unique challenges. The application of comprehensive assays in the diagnostic setting involves multiple disciplines and a sequencing pipeline that moves smoothly from sample intake through the completion of a molecular diagnostic ‘report’ with pathology sign-off. My lecture will focus on specific details of our current diagnostic approaches, how they are presented to the signing pathologist, and how we maintain the integrity and clinical validity of the variants we report.
S27
ENSURING CLINICAL VALIDITY – MODERNISING GENETIC TESTING SERVICES Karin S. Kassahn1, Hamish S. Scott1,2 and Janice M. Fletcher1,3 1Genetic and Molecular Pathology, SA Pathology, 2Centre for Cancer Biology, SA Pathology, and 3School of Paediatrics and Reproductive Health, Faculty of Health Sciences, The University of Adelaide, Adelaide, SA, Australia Genomic testing using gene panels and whole-exome sequence data are becoming increasingly attractive options to support the clinical management of patients. Whole-exome sequencing to diagnose rare genetic disorders in paediatrics offers a cost-effective and efficient first screen for cases where there is no obvious clinical diagnosis, thus fundamentally shifting medical service provision paradigms. In other areas of medicine, the implementation of targeted gene panels using next generation sequencing is associated with improved diagnosis rate and faster time to diagnosis. Successful integration of genomic testing into the patient management pathway requires updating of the traditional models of test provision. Important factors to be considered include: who should qualify for what genomic test, payment for testing, the appropriate referral mechanism, and how to effectively communicate the results and limitations of genomic tests to patients and clinicians. We discuss strategies and give examples from implementing gene panels and whole-exome sequencing in clinical diagnostics to support the management of patients in South Australia. The next few years are likely to be an exciting and challenging time in genomic test provision in Australia, as these new platforms establish themselves as part of routine medical care and different stakeholders (local, international, direct-to-consumer) define their share of the clinical sequencing market.
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.