Platform Presentation Assessing copy number from exome ... · An overall positive detection rate of...

2014 ICCG Meeting Abstracts I 1

Platform Presentation

Assessing copy number from exome sequencing and exome array CGH based on CNV spectrum in a large clinical cohort

Eden Haverfield1, Kyle Retterer1, Julie Scuffins1, Daniel Schmidt1, Rachel Lewis1, Daniel Pineda Alvarez1, Amanda Stafford1, Lindsay Schmidt1, Stephanie Warren1, Ludmila Matyakhina1, Jeanne Meck1, Swaroop Aradhya1

1GeneDx, Gaithersburg, MD

Detection of copy number is an important consideration in testing for a wide variety of genetic disorders. Array CGH has significantly enabled the interrogation of disease genes for copy number mutations. More recently, various algorithms to analyze whole exome sequencing have made it possible to extract copy number information from sequence read depth. Here we present data from exon-focused arrays used for testing 14,022 individuals for copy number mutations in one or more genes. An overall positive detection rate of 2.4% was observed among these individuals. The mutations included 318 deletions and 25 duplications. Only one or two exons were deleted in 40% of mutations, highlighting the necessity for high-resolution in any copy number assay. Separately, we also describe data from 14,000 individuals referred for whole genome chromosomal microarray analysis. We identified 3,345 CNVs affecting a single gene, representing 18% of all CNVs identified by whole genome CMA. Together, these data provide a deep picture of the types and sizes of intragenic copy number mutations in disease genes in single-gene and whole genome screening contexts. Based on these results, we have developed a bioinformatics algorithm to detect deletions and duplications in whole exome sequence data and show that it can detect intragenic mutations including three or more exons as well as submicroscopic and large cytogenetic imbalances. In addition, we have also validated a novel whole exome array that can be used to test for single gene intragenic copy number mutations or as an ultimate cytogenetic whole genome chromosomal microarray screen. This array complements exome sequencing to unambiguously identify intragenic mutations and can also detect single-exon changes. The data presented here illustrate the spectrum of intragenic copy number mutations and provide the foundation for the next advancement in copy number analysis approaches through whole exome sequencing and whole exome array.



Towards comprehensive clinical exome sequencing: Inclusive testing of disease genes with high homology

Diana Mandelker1, Arunkanth Ankala2, Elizabeth Duffy3, Rimma Shakhbatyan3, Kristin Gibson4, Sami Amr3, Heidi L. Rehm1,3, Avni Santani4, Matthew Lebo1,3, Madhuri Hegde2, Birgit Funke1,3

1Department of Pathology, Harvard Medical School, Boston, MA 2Emory Genetics Laboratory, Emory University School of Medicine, Atlanta, GA 3Laboratory for Molecular Medicine, Cambridge, MA 4Department of Pathology and Laboratory Medicine; Children’s Hospital of Philadelphia, Philadelphia, PA Exome sequencing (ES) is being implemented in clinical laboratories to accelerate the diagnosis of genetic disease. While the goal of clinical ES is the complete analysis of all medically relevant genes, the presence of sequences with high homology to functional genes hinders this effort. Homologous sequences can pose analytical challenges for medical NGS, as unique read mapping may be impossible, increasing the risk for both false negative and false positive variant calls. To enable accurate and comprehensive genetic testing, it is imperative to accurately define medically relevant genes whose analyses are complicated by pseudogene interference and to devise alternate testing strategies for those genes.

By parsing databases containing gene-disease associations, we assembled a list of 4631 genes with possible or likely clinical importance, termed the “Medical Exome.” A combination of sliding window homology and empirical mapping quality analysis identified 132 genes with regions of homology that preclude accurate analysis by NGS technologies. We have added medical and technical annotations, and developed a catalog of curated genes with homology issues to alert clinical labs using NGS of the difficulty in accurately calling variants in these regions.

Our ultimate goal is to develop a multiplexed assay enabling simultaneous analysis of multiple genes with homology issues in a way that is compatible with clinical diagnostic workflows. Towards this goal, we chose the Stereocilin (STRC) gene, which is 99.6% identical to a pseudogene, as our model for establishing a framework for the analysis of such genes. We developed a long range PCR assay and showed by both Sanger and MiSeq analysis that this eliminated pseudogene contamination. Comparison to standard capture based NGS revealed that 18% (2/11) of SNV calls were false positive due to pseudogene interference, a situation with potential dire consequences in a clinical setting. This assay increased the clinical sensitivity of our NGS non-syndromic hearing loss test (70 genes) from 20.4% to 31.6%, further underscoring the importance of interrogating such genes.

In summary, we have developed a resource for use by the genetics community to avoid pitfalls surrounding homologous genes. Additionally, we have demonstrated both an approach to and the clinical importance of analyzing these technically challenging genes, and propose a future multiplexed assay for medically important genes affected by homology.



Phevor combines phenotype and genotype to expand molecular diagnostics beyond disease variant databases

Marc V. Singleton1, Karen Eilbeck2,3, Martin G. Reese4, Mark Yandell1,2

1Department of Human Genetics, Eccles Institute of Human Genetics, University of Utah School of Medicine, Salt Lake City, UT 84112, USA 2Utah Science Technology and Research Center for Genetic Discovery, University of Utah, Salt Lake City, UT 84112, USA 3Department of Biomedical Informatics, University of Utah, Salt Lake City, UT 84112, USA 4Omicia Inc., 1625 Clay Street, Oakland, CA 94612, USA Successful molecular diagnosis using an exome sequence hinges on the association of a damaging variant to the patient’s phenotype. Unfortunately many clinical scenarios (e.g. single affected or small nuclear families) have little power to confidently identify damaging-alleles using sequence data alone. Today’s diagnostic tools are simply underpowered for accurate diagnosis in these situations, limiting successful diagnoses. In response, clinical genetics are forced to rely on candidate-gene and variant lists to limit the search space. Despite their practical utility, these lists suffer from inherent and significant limitations. Impact of false negatives on diagnostic accuracy is considerable because candidate genes/variants lists are assembled ad hoc, choosing alleles based upon expert knowledge. Alleles not in the list are not considered—ending hope for novel discoveries. Rational alternatives to ad hoc assemblages of candidate lists are thus badly needed. In response, we created Phevor, the Phenotype Driven Variant Ontological Re-ranking tool. Phevor works by combining knowledge resident in biomedical ontologies, like the Human Phenotype and Gene Ontologies, with the outputs of variant-prioritization tools such as SIFT, GERP+, Annovar and VAAST. This enables Phevor to accurately reprioritize candidates identified by 3rd party variant-prioritization tools in light of knowledge found in the ontologies, effectively bypassing the need for candidate-gene/variant lists. Phevor differs from tools such as Phenomizer and sSAGA as it does not postulate a set of fixed associations between genes and phenotypes. Rather, Phevor dynamically integrates knowledge resident in multiple bio-ontologies into the prioritization process. This enables Phevor to improve diagnostic accuracy for established diseases and previously undescribed or atypical phenotypes. Phevor was benchmarked in recently published work by spiking known disease-alleles into otherwise healthy exomes. Using the phenotype of the known disease, and the variant-prioritizing tool VAAST (Variant Annotation, Analysis and Search Tool), Phevor is able to rank 100% of the known alleles in the top 10 and 80% as the top candidate. Phevor is currently used to diagnose cases as part the Utah Genome Project. Benchmarking results demonstrate recent efforts to incorporate gene expression, pathway and drug interactions into Phevor for still more accurate diagnosis of disease-alleles, especially for common diseases.



The Current State of Panel Testing and Mechanisms for Phenotypic Data Collection within the Genetic Testing Industry

Judsen D. Schneider1, Taylor Murphy1, Jon Staples1, Marshall Cottrell1, Blake Blackshear1, Gillian W. Hooker1

1Department of BioInformatics, NextGxDx, Inc. Nashville, TN

The rapidly expanding and dynamic clinical genetic testing market is becoming increasingly challenging to navigate from any number of entry points. On the ordering side, busy clinicians are faced with more testing and laboratory options than ever before. On the laboratory side, as tests become more generalized (e.g. expanded gene panels and exome sequencing), there need to be specific, standardized processes for collection of patient data to aid in interpretation. The recent development of a standardized ontology for human phenotyping by the International Consortium of Human Phenotype Terminology (ICHPT) represents a significant step forward. However, practical mechanisms for the collection and transfer of this information from clinicians to laboratories and between collaborating laboratories are limited.

Because of this and other issues related to a paper-based workflow, NextGxDx has developed a system to facilitate communication and connectivity in the genetic testing marketplace, providing laboratories and clinicians with a comprehensive and up-to-date information resource, online ordering system, and result delivery tool. As a consequence of this unique positioning within the industry, NextGxDx is ideally positioned to support consortium-based research endeavors seeking to incorporate digitized and standardized clinical information.

To aid in the development of a universal system for collecting phenotypic information, we analyzed the existing models of patient data collection used by clinical reference laboratories for panel and exome-based testing. From our database of CLIA-certified genetic testing laboratories, as of April 7th, 2014, there are over 19,000 genetic testing products currently available to the US market, including 2,143 panels (>1 analyte, enzyme or gene) and 15 clinical exome products. Only a subset of products require clinical information on their requisition form. Data is collected in a range of formats, in many cases using non-standardized terminology, and through multiple mechanisms (ranging from custom digital solutions to consortium-developed forms). Also, the depth of information requested ranges from none to the patient’s entire medical record. We will present an overview of the industry and the results of this study to facilitate a broader discussion about a functional solution for the collection and delivery of phenotypic data.



Enhancing Phenotypic Data Collection Through Patient Entered Data

Bethanny Smith-Packard, Stephen Martin, W. Andrew Faucett, Simons Variations in Individuals Project

Geisinger Health System, Danville, PA

The goal of the Simons Variations in Individuals Project (Simons VIP) is to characterize the natural history of individuals with copy number variants (CNVs) related to autism (e.g., del 16p11.2) by collecting phenotypic information through evaluations of probands and family controls. Since September 2010, a website (www.simonsvipconnect.org) has served as a patient registry and has been used as a novel method of online recruitment and community support and engagement. Initially, the Simons VIP study focused on in-person evaluations at one of the five collaborating medical centers throughout the US, while the website was used for recruitment and community engagement. The website has provided a successful model of subject recruitment and retention; in three years the community grew to over 800 members, recruiting over 400 probands from 243 families, with an average of 6 new registrants per week.

In February 2014, “Phase 2” of the study was launched, utilizing remote (online, mail, phone) assessments to augment the collection of phenotypic information. To accommodate the transition from in-person evaluations to remote assessments, the website was re-designed to serve as a portal to online research participation, while maintaining its function as a community support and resource center. In developing website content, a priority was to maximize patient-friendliness and accessibility to the general population. Now, as part of registration to the online community, families opt in or out of research participation by choosing to be a “research-community member” or a “community-only member,” respectively. Participating families undergo online consent and complete online research surveys that currently average 2 hours per family and may increase to about 10 hours as new assessment tools are developed. Longitudinal reassessment is planned at designated intervals based on age and carrier status. In the first two months of Phase 2, 31 out of 243 Phase 1 families have re-enrolled to participate in Phase 2; 66 individuals have completed online surveys. A plan is in place to re-enroll the remaining Phase 1 families, while also recruiting new families, as well as those families not able to participate in Phase 1 due to travel limitations. The success of the novel online research model being utilized in Phase 2 of Simons VIP demonstrates that families are willing to provide detailed phenotype information online, increasing the potential amount of data that can be collected.


1. Understanding and sharing ethnicity specific data: BRCA Testing in India as an example

Urvashi Bahadur1, Dana L. Abramovitz2, Vamsi Veeramachaneni1, and Ramesh Hariharan1

1Strand Life Sciences, Bangalore, India 2Strand Genomics, San Francisco, CA The correlation between variants in the BRCA1 and BRCA2 genes and susceptibility to breast and ovarian cancer is well known. However obtaining population and ethnicity specific information about susceptibility is a key challenge. Although the prominent BRCAnalysis test by Myriad is available worldwide, variant-level data is not freely available. Strand Centers for Genomics and Personalized Medicine, based in Bangalore India, perform a 94 gene based hereditary cancer predisposition test in which BRCA1 and BRCA2 are included. Over the last year we have collected nearly a hundred samples from individuals of Indian descent and analyzed them for cancer susceptibility. Using StrandOmics, Strand’s clinical interpretation and reporting platform, we are able to classify and report on various BRCA variants. StrandOmics also allows us to better assess the significance of identified variants and their relative prevalence by ethnicity by comparing them with other identified variants in StrandOmics’ Pooled Patient DataBase (PPDB). The StrandOmics PPDB also facilitates sharing of the BRCA variant data across internal labs, other facilities using StrandOmics, and the broader medical community. Sharing of more BRCA variants using StrandOmics will enable a better understanding of the genotype-phenotype correlation in these important genes.


2. Top 10 Misconceptions of Clinical NGS Sequencing

Richard Chen, Mark Pratt, Deanna M. Church, Sarah Garcia, Michael Clark, Jason Harris, Gabor Bartha, Anil Patwardhan, Stephen Chervitz, John West

Personalis Inc, Menlo Park, CA

Whole exome and genome sequencing are increasingly used for clinical diagnosis of rare genetic syndromes yet clinicians often overlook critical issues with next-generation sequencing (NGS) that can impact diagnostic accuracy. This talk will seek to raise, and dispel some of the important misconceptions regarding accuracy and the clinical application of NGS. These common misconceptions include: (1) NGS gold standards are gold (2) Exomes cover the whole exome (3) Standard exomes are sufficient for clinical use (4) Whole genomes are better than exomes for clinical applications (5) Average depth of sequencing is an adequate predictor of quality and accuracy (6) Coverage gaps in genes can be fixed by simply sequencing to higher depth (7) 10X coverage is enough to call variants (8) The human reference used has minimal clinical diagnostic impact (9) The sequencing, alignment/variant calling, and interpretation steps of the clinical NGS workflow can be optimized independently of each other (10) There is sufficient data in public databases for clinical variant interpretation. This talk will walk through each of these issues and give data and examples that counter these perceptions.


3. Impacts of updating the reference assembly on genome interpretation Deanna M. Church, Jason Harris, Stephen Chervitz, Gabor Bartha, Shujun Luo, Mirian Karbelashvili, Ming Li, Amy Huang, Parin Sripakdeevong, Scott Kirk, Michael Clark, Sarah Garcia, Mark Pratt, John West and Richard Chen Personalis Inc., Menlo Park CA While the sequencing of human genomes has become a routine procedure, the interpretation of these genomes is challenging and reliant upon the reference assembly. The Genome Reference Consortium (GRC) released an updated version of the reference assembly (GRCh38) in late 2013. GRCh38 contains many updates that have a profound effect on genome interpretation. These updates include the addition of several megabases of human specific sequences as well as the targeted correction of many small regions that affect the interpretation of clinically relevant genes (e.g. SHANK1, SLC46A1, POU3F4). The adoption of this reference requires both the development of more sophisticated analysis pipelines and the re-evaluation of annotation data that has accumulated on older assembly versions. We are addressing both of these fronts as we move to adopt GRCh38. We are developing a new analysis pipeline that will initially take advantage of the fix patches released for GRCh37. This allows us to investigate strategies to move beyond using just the chromosome sequences and take advantage of the full assembly. We can also take advantage of many corrections without having to remap the annotation information used for variant interpretation. Additionally, we are modifying the pipeline to always evaluate known locations of medical relevance. While many such regions are corrected in GRCh38 many sites remain. Remapping of the roughly 1 million rare sites found in GRCh37 and corrected in the Personalis Major Allele Reference shows that only 16,000 of these sites have an allele change, suggesting that many rare sites remain in GRCh38. In order to identify medically relevant variants it is critical to compare observed variant data to variant and gene annotations from archival databases such as ClinVar and OMIM. However, while mapping the data onto the new coordinate system is relatively trivial, interpretation this data is not always straightforward. For example, mapping of the roughly 55,000 sites in ClinVar (as of Apr. 2014) finds that the vast majority of these sites have a location in GRCh38 but approximately 100 of these sites map to regions that are collapsed in GRCh37, suggesting that paralogous sequences have been added to GRCh38. Interpretation of variants in these regions will take additional review. Identifying these problematic annotations is a critical part of our strategy for moving towards the new reference assembly.


4. Complete Exon Coverage of the BRCA 1 and 2 Genes Using a Massively-Parallel Singleplex PCR Targeted Enrichment for NGS Library Preparation

Jude Dunne, Max Sanchez, Alain Mir, Scott Silveria, Masy Leong, Glenn Hein, Sangeetha Anandakrishnan, Alan Chang, Monika Tomczyk, Elaine Wong-Ho and Gianluca Roma

WaferGen Biosystems, Fremont, CA

The main challenge for clinical targeted next-generation sequencing methods is obtaining complete and uniform coverage of all target regions. Some popular methods for target enrichment rely on lengthy and inefficient hybrid capture resulting in lower coverage and higher off target reads. To address these challenges, WaferGen Biosystems has developed the Seq-Ready™ TE DNA panels for the preparation of genomic DNA libraries that can be completed in less than 4 hours with less than 45 min of hands on time. The Seq-Ready™ TE Custom DNA Panels allows researchers to create custom targeted gene panels of up to 2.5 Mb of cumulative genomic regions for excellent design flexibility. The Seq-Ready™ TE BRCA 1/2 Panel target 100% of the coding regions the BRCA 1 and BRCA2 tumor suppressor genes. Unlike traditional multiplex PCR assay designs that often require multiple rounds of highly empirical optimization and careful primer pooling, the SmartChip™ massively-parallel singleplex technology eliminates the need for lengthy primer redesign and thereby accelerates the assay development process. The Seq-Ready TE Custom DNA Panels allow researchers to obtain higher first time in-silico design (typically > 98% of all ROI) and higher amplicon conversion rates (typically > 95%). Sequencing results of 24 samples processed on a single SmartChip using the Seq-Ready™ TE BRCA 1/2 Panel showed 100% coverage at >100x for all targeted regions. With high coverage and high on-target rates, clinical researchers can utilize the Seq-Ready TE DNA panels for reliable and cost-effect target enrichment.


5. MedSavant: A high-performance search engine for genetic variants

Marc Fiume1, Orion Buske1, James Vlasblom1, Eric Smith1, Andrew Brook1, Khushi Chachcha2, Sergiu Dumitriu1, Christian R. Marshall3, Kym M. Boycott4, Marta Girdea1, Peter Ray3, Gary Bader5, Michael Brudno1,3 1University of Toronto, Toronto, Ontario, M5S 1A1, Canada 2University of British Columbia, Vancouver, British Columbia, V6T 1Z4, Canada 3The Hospital for Sick Children, Toronto, Ontario, M5G 1X8, Canada 4Children’s Hospital Ontario Research Institute, University of Ottawa, Ottawa, Ontario, K1N 6N5, Canada 5The Donnelly Centre, Toronto, Ontario, M5S 3E1, Canada Recent advancements in DNA sequencing technologies have economized the sequencing of individual genomes, creating potential to improve diagnostics and treatment for patients affected by genetic diseases. A significant challenge in utilizing these technologies in the clinical setting has been in the implementation of data analysis strategies that can be used to easily and efficiently identify causative genetic mutations from the large number of variants discovered through sequencing. A common approach is to annotate and iteratively filter potentially causal genetic mutations using ad-hoc combinations of computational scripts and their parameters; however, this method necessitates informatics expertise, is slow, non-interactive, and does not scale to the extent needed for clinical and other large-scale sequencing applications.To facilitate the discovery of causative genetic mutations, we have developed MedSavant, an integrated solution for the storage, annotation, filtration, prioritization, and visual inspection of variants. It is entirely graphical, interactive, and scalable to manage datasets generated by large-scale sequencing projects. To accomplish this, the system employs big data analytics technologies optimized for genomic datasets that are capable of delivering the results of complex dynamic queries nearly instantaneously while using significantly less storage resources compared to the standard flat file equivalent.

The MedSavant platform was designed for use in large-scale research and clinical genome sequencing projects. We have created two MedSavant installations to demonstrate its use in these respective applications. The first installation contains over 130,000,000 unique variant entries from half of the 1000 Genomes Project individuals. This installation is publically accessible via an online portal. The second installation contains clinical data from 425 individuals from the Care for Rare Consortium, a set of projects whose aim is to discover the etiology of rare genetic disorders. Using MedSavant equipped with the internally-developed Mendel App, we independently discovered the causal gene in 15 tested FORGE Projects.


6. The Clinical Exome: Personalis’ Experience Using an Enhanced Exome and Genome-wide Structural Variant Detection for the Diagnosis of Diseases of Unknown Genetic Etiology

Sarah Garcia, Jeanie Tirch, Jason Harris, Anil Patwardhan, Stephen Chervitz, Deanna M. Church, Michael Clark, Gemma Chandratillake, Mark Pratt, John West, Richard Chen

Personalis Inc, Menlo Park, CA

Despite the theoretical power of exome sequencing, the realized diagnostic yield of clinical exome testing remains relatively low, ~25%. False negatives, which may account for a significant proportion of unsolved cases, stem from both technical and analytical challenges- including inadequate sequencing coverage, undetected variation (e.g. structural variation), non-optimized prioritization of variants, limited understanding of the impact of genetic variation on phenotype. The ACE (Accuracy and Content Enhanced) Clinical Exome addresses these challenges and aims to increase diagnostic yield through the use of ACE sequencing, extensive annotation, and a suite of phenotype-based analytics.

To date, we have used the ACE Clinical Exome to analyze 55 cases of undiagnosed disease. An additional 70 cases are currently in review. Variants of interest were reported in 31 (56%) cases, with the majority having causative/likely causative variants in genes known to cause phenotype. A few cases had variants in genes partially explanatory of phenotype (2 cases), variants in candidate genes for phenotype (2 cases), or a single variant identified in a gene known to cause recessive disease (3 cases). Two causative SVs were identified- a large deletion and a previously reported single exon deletion. Ten cases were sequenced with both ACE Clinical Exome and high-depth whole genome. No variants of interest were identified in whole genomes that were not also detected by ACE Exome. Qualitatively, we did not see diagnostic yield differences in proband-only vs. trio analyses.

Of the genes for which we reported variation, the majority showed deficits in sequencing coverage on standard exome platforms compared to supplementation with ACE sequencing. At a standard of 25X mean coverage, standard exome sequencing misses 4-5% of coding bases in reported genes while only 0.7% of bases were covered at <25X using ACE sequencing.

We demonstrate the ACE Clinical Exome’s diagnostic yield in cases of unknown genetic etiology. The ACE sequencing approach improved sequence coverage in genes with reported variation and detected SVs causative of disease. Due to prior aCGH testing (44% of cases), our pilot set was depleted for cases likely to be explained by large SVs, and thus diagnostic yield may be further improved in an unselected patient cohort. An observational case study is underway to assess the diagnostic performance of the ACE Clinical Exome in a consecutively referred patient population.


7. dbVar: a growing catalog of structural variation and clinical significance

Tim Hefferon1, John Lopez1, John Garner1, Deanna Church1, Lon Phan1

1NIH, NCBI, Bethesda, MD

dbVar is a public database of genomic structural variation. It is one in a suite of variation databases maintained at the National Center for Biotechnology Information (NCBI). dbVar and its sister database DGVa (at EBI) collect and consolidate structural variation submissions to create mirrored resources that capture and accurately represent the spectrum of observed structural variation. This joint catalog contains almost 4 million SV from 120 studies in 16 organisms. The studies include data from human consisting of genome-wide surveys, comparisons of case and control populations, tumor versus matched control samples, and large curated studies with data derived from multiple sources. Together dbVar and DGVa represent the most comprehensive source of SV data in the world, and include data originating from the 1000 Genomes Project (studies estd59 and estd199), The Wellcome Trust Sanger Institute Mouse Genomes Project (estd118), the Catalogue of Somatic Mutations in Cancer (COSMIC; estd192), and numerous clinical genetics studies. In collaboration with ClinVar (http://www.ncbi.nlm.nih.gov/clinvar/), dbVar also curates structural variants that are accompanied by assertions of clinical significance; users can follow direct links to corresponding ClinVar reports where they can find detailed information describing the asserted relationships between human variations and phenotypes.


8. StrandOmics: A powerful Interpretation and Reporting Tool for Analysis and Sharing of NGS-based Test Data

Shuba Krishna, Smita Agarwal, Vamsi Veeramachaneni, and Ramesh Hariharan

Strand Life Sciences, Bangalore, India

Strandomics is a clinical interpretation and reporting tool that expedites variant interpretation using an in-house curated database combined with popular databases like HGMD, ClinVar and dbSNP as well as various bioinformatics tools. Individual or multiple samples in a family can be analyzed together in the tool and the variants, ordered by pathogenicity, are called out in the genes of interest. The importance of a variant in the phenotype can be ascertained by studying if the variant segregates with a phenotype in similarly affected individuals, or, if it is absent in normal individuals or individuals with an unrelated phenotype. These comparisons are greatly enhanced through the use of the pooled patient database (PPDB), which is a repository of all samples, affected and unaffected, and can enable data-sharing across various facilities without compromising data privacy.

At Strand Centers for Genomics and Personalized Medicine, based in Bangalore India, we have helped many families with diagnosis and risk predictions for various cardiac disorders, opthalmological disorders, rare inherited disorders and cancers etc. through NGS based sequencing tests. The interpretation and reporting of the sequencing data was done using StrandOmics. Here we present a test case of a 5-member family where the proband suffered from premature coronary artery disease and wanted a risk assessment for his unaffected family members. Premature coronary artery disease (pCAD) is almost an epidemic in India, with the disease manifesting as early as the thirties. Through our curation effort, we created a test including fifty-two genes likely to be important in pCAD in the South Asian population. Through a single analysis, we were able to identify two variants, one each in the SELP and ABCA1 gene in the proband likely to be causative for his condition. Of his two children, both had inherited one of the variants from him, but not both and were thus predicted to be at some risk albeit lower than the proband of developing pCAD. The wife and the cousin who were also analysed did not carry these or any other likely causative variants for pCAD in the 52 genes analysed. Thus using StrandOmics, we were quickly able to narrow down the possible causative variants responsible for the probands disease as well as provide risk analysis for his unaffected family members.


9. Homocystinuria caused by a novel pathogenic variant of CBS diagnosed by whole exome sequencing after CMA testing.

Joel A. Lefferts1, Mark A. Cervinski1, Mary Beth P. Dinulos1, Torrey L. Gallagher1, Emmeline Z. Liu1, Elizabeth I. Reader1, Heather B. Steinmetz1, Scott A. Turner1, Stephanie E. Vallee1, Marco Tulio Martinez Membreño2, Marco Tulio Martinez2, Roberto Elvir Zelaya3, Suzanne P. Burgos4, Derek Sven Stenquist5, Richard Chen6, Sarah Garcia6, Deanna Church6, Jeanie Tirch6, Amy Huang6, Linda Kennedy1, Peter Mason1, Dean Seibert1 and Gregory J. Tsongalis1

1Dartmouth-Hitchcock Medical Center, Lebanon, NH and Geisel School of Medicine at Dartmouth, Hanover, NH. 2Liga Contra el Cáncer, San Pedro Sula, Honduras. 3Hospital Cemesa, San Pedro Sula, Honduras. 4Gifford Medical Center, Randolph, VT. 5Harvard Medical School, Boston, MA. 6Personalis, Menlo Park, CA. Introduction: Homocystinuria is an autosomal recessive metabolic disorder in which elevated concentrations of methionine and homocysteine are cause by a defect in the conversion of homocysteine to cysteine. The defect caused by mutations in the cystathionine beta-synthase gene (CBS) results in a variable phenotype often including developmental delay, intellectual disability, skeletal abnormalities, ocular defects, osteoporosis and thromboembolism. The clinical impact of this disorder is often decreased by dietary modification. We present the case of a family from an isolated Central American village with two children similarly affected by intellectual disability and dysmorphic features of unknown etiology. Methods: DNA samples were extracted from buccal swabs from two affected siblings (11 yo and 14 yo) and their mother. Chromosome microarray (CMA) analysis was performed using the CytoScan® HD Array (Affymetrix, Santa Clara, CA). Augmented whole exome sequencing, with identification of the putatively causal deletion was performed on DNA from the oldest sibling (ACE clinical exome, Personalis). Targeted sequencing of the identified locus was performed by standard Sanger sequencing. Results: CMA performed as first-tier testing on the two siblings was uninformative with respect to copy number variation but did detect significant LCSHs covering 25-30% of the autosomes for both siblings. A search for genes associated with autosomal recessive disease in the LCSH regions shared by both siblings resulted in 218 genes. Whole exome sequencing of the older sibling identified homozygosity for a novel pathogenic variant in CBS. A diagnosis of homocystinuria was further confirmed in this patient by detection of elevated blood homocysteine concentration. Sanger sequencing detected the same mutation in the other sibling (homozygous) and in the mother (heterozygous). Conclusions: This report of undiagnosed homocystinuria highlights several relevant reminders for clinicians as we move forward in this era of global medicine and genomic testing. Genetic diseases that are part of many newborn screening efforts should not be overlooked when treating patients without access to standard medical care. SNP-based CMA testing can help identify candidate genes when an unknown autosomal recessive disorder is suspected but may be less effective in the case of extensive LCSHs and often requires extensive phenotypic information to produce a manageable list of candidate genes.


10. Meeting the Standards of Traditional Genetic Testing with Next Generation Sequencing

Stephen Lincoln1, Allison Kurian2, Leif Ellisen3, Yuya Kobayashi1, Michael Anderson1, Michele Gabree3, Andrea Desmond3, Geoffrey Nilsen1, Kevin Jacobs1, Martin Powers1, Swaroop Aradhya1, James Ford2

1Invitae Corporation, 2Stanford University, 3Massachusetts General Hospital Next-Generation Sequencing (NGS) technology is gaining acceptance in clinical practice, although some questions remain about its diagnostic performance. Sensitivity for “hard” variant classes (indels, complex sequence variants, and small and large del/dup events) and overall specificity are reasonable concerns, and most clinical NGS labs (including ours) orthogonally confirm reported variants, adding to expense and time. Separately, concern is also raised about the challenges in interpretation and clinical use of expanded genetic information which is enabled by NGS.

We used NGS to test over 1000 germ-line DNA samples from two major clinical centers for sequence changes and del/dup events in a panel of 29 hereditary cancer genes. These individuals were also independently tested for BRCA1/2, and sometimes other cancer risk genes, using traditional methods such as Sanger sequencing and QPCR. We also gathered a set of over 100 reference samples with traditional data for various genes and we particularly attempted to enrich this set for known examples of “hard” variants for NGS. Finally, we also included 7 samples for which we could derive high-quality genome-wide data, including the NIST “Genome in a Bottle” reference NA12878. Comparison of the NGS data for these samples to the reference data allowed us to measure both sensitivity and specificity of our NGS methods. Because the independent data can sometimes be incomplete, samples with discordant calls were sent to a third party lab for resolution.

For the 712 samples thoroughly analyzed to date, NGS detected all 2,374 previously known variants (pathogenic and otherwise) in the reference data for the assayed genes. Among these were 69 examples of particularly “hard” variants including (a) indels from 10 to hundreds of base-pairs, (b) del/dup events as small as single exons and as large as entire chromosomes, and (c) certain homopolymer associated pathogenic variants. We saw no false positives in the 2200 individual gene tests effectively represented in this set. More recent bioinformatics analyses of these and other data suggests various metrics of both regional genomic context and sample-specific NGS raw data that may reliably indicate sites where NGS results are highly accurate, as opposed to those cases where additional scrutiny is required in clinical reporting to avoid false positives and false negatives.

We have begun the process of submitting these data to ClinVar, including clinical assessments following ACMG guidelines, making comparisons between labs possible for these variants. We hope that such data from our lab and from others can help inform longer term views in the laboratory community of how NGS and traditional methods can best be used, independently and together, to achieve the needed analytic and clinical performance for NGS-based testing.


11. Sensitive somatic mutation detection in cell-free circulating DNA with qBiomarker somatic mutation PCR array coupled with preamplification Samuel Long and Eric Lader QIAGEN Center of Excellence in Biological Content, Frederick, MD, USA Cell-free circulating DNA (cfDNA) as a non-invasive, liquid biopsy has recently been exploited for use in early cancer detection, patient stratification, prognosis, disease/treatment monitoring, prenatal test, as well as molecular epidemiology studies. cfDNA provides a unique window into a cancer patient’s molecular landscape. Compared to the static sampling of DNA from an archival/FFPE tumor, cfDNA provides real time mutational information of primary and metastatic tumors, which can be used to facilitate implementation of treatment decision and monitoring. cfDNA mutation analysis also has the advantage of allowing interrogation of the intra-patient mutation heterogeneity. The challenges for mutation analysis in cfDNA include the low concentration of cfDNA in a patient’s plasma or serum and the potentially low level of mutations in this low concentration of cfDNA. In addition, the fragmented nature of some cfDNA samples can further reduce the effective input in PCR-based detection applications. QIAGEN has developed and optimized a PCR-based, targeted preamplification solution that enriches the target regions of interest in cfDNA by 4000 fold, therefore dramatically increasing the effective template input for downstream mutation analysis using qBiomarker somatic mutation assays or arrays. Preamplification can be performed either in singleplex PCR to allow single mutation detection, or in a highly multiplex PCR reaction (up to 400 plex) to allow mutation profiling of up to 1217 frequently occurring cancer hot spot mutations occurring in 163 genes. In this study, targeted preamplification paired with qBiomarker mutation PCR array was used to detect serum or plasma cfDNA mutations that were known to be present in the corresponding lung or colon cancer patients’ primary tumor tissues. In 3 out of 5 cases, the same mutations characterized in the solid tumors were present at 0.2% to 0.6% level in cfDNA. In the 2 non-concordant cases, the mutations appeared to be absent from the cfDNA or below the mutation assay detection limit. qBiomarker mutation PCR array screening also identified 2 additional somatic mutations from the cfDNA. These mutations were previously not examined in the solid tumor samples. An analysis of an additional 15 cfDNA from previously un-genotyped colon, lung, breast and endometrial cancer patient serum samples detected 6 somatic mutations at levels ranging from 0.06% to 0.4%. We conclude that the pairing of targeted preamplification and qBiomarker mutation PCR arrays/assays provides a cost-effective translational research tool to detect cancer mutations earlier and more sensitively in cfDNA. The method has the potential to monitor 1217 mutations in 163 genes from the same cfDNA sample. The applications presented here are for research use only. Not for use in diagnostic procedures.


12. A clinically driven approach to the implementation of whole exome sequencing in clinical care across multiple institutions - the Melbourne Genomics Health Alliance

I. Macciocca1, N. Thorne1, A. Boussioutas3,4, P. A. James3,4, P. Kwan3,4, A. Oshlack3,5, A.W. Roberts2,3,4, M. Ryan3,5,6, G. Taylor3, S. M. White3,5,7, C. Gaff1

1Melbourne Genomics Health Alliance, Parkville, Melbourne, Australia 2Walter and Eliza Hall Institute, Melbourne, Australia 3University of Melbourne, Melbourne, Australia 4Royal Melbourne Hospital, Melbourne, Australia 5Murdoch Childrens Research Institute, Melbourne, Australia 6Royal Children's Hospital, Melbourne, Australia 7Victorian Clinical Genetics Services, Melbourne, Australia

Unprecedented advances in sequencing technologies have brought whole exome sequencing (WES) to the clinic sooner than many anticipated. Single institution, laboratory-led, clinical WES in specific patient groups has been reported but challenges remain in establishing a unified approach for the diverse clinical needs of genomics technologies across multiple conditions and health services.

The Melbourne Genomics Health Alliance, a collaboration of six research organizations and hospitals in Melbourne Australia has developed a clinician-led approach to WES that can be applied to multiple patient groups in its Alliance hospitals. Expert clinicians from the two Alliance hospitals selected five patient groups – Charcot-Marie-Tooth disease, childhood syndromes, acute myeloid leukaemia, focal epilepsy and familial colorectal cancer - that would benefit from WES as part of a demonstration project using a common bioinformatic pipeline with targeted analysis of genes associated with each of the conditions. Clinicians’ preferences regarding genes to be targeted for analysis, pipeline output and variant prioritization and calling were determined and incorporated into the pipeline design. A bioinformatics advisory committee, which included clinician representation, developed the common pipeline based on these preferences and taking into account the potential to incorporate other disease groups in the future. A common reporting format, informed by clinician and patient preferences, is in development. The unique features of the Melbourne Genomics approach to clinical WES will be presented.


13. Supporting the MedSeq Project through a systematic approach to genomic interpretation Kalotina Machini1-3, Rimma Shakhbatyan3, Heather McLaughlin3-5, Ozge Ceyhan Birsoy3-5, Danielle Metterville3, Robert Green1,2, Matt Lebo3-5, Heidi L. Rehm3-5

1Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA 2Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts, USA 3Laboratory for Molecular Medicine, Partners Personalized Medicine, Cambridge, Massachusetts, USA 4Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA 5Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts, USA The greatest obstacle in comprehensive whole genome sequencing (WGS) for clinical use is the difficulty in systematically interpreting the enormous amount of data generated. This includes the interpretation of previously published as well as novel genetic variants. Within the framework of the MedSeq Project, we are curating a dataset of genes and variants to efficiently support WGS annotation and share among laboratories. 100 participants, half healthy and half with cardiomyopathy, are undergoing WGS. Sequencing is performed at Illumina’s CLIA-certified laboratory and alignment, variant calling, annotation and variant review are performed at the Laboratory for Molecular Medicine. The annotated variants are filtered to identify: i) variants with a minor allele frequency (MAF) <5% and classified as DM or DM? in the Human Genome Mutation Database (HGMD); ii) nonsense, frameshift, and canonical splice-site (+/-1,2) variants with a MAF <1% from the medical exome gene list. The evaluation of potential Mendelian disease-causing variants involves analysis of multiple lines of evidence including allele frequency, computational prediction tools and review of genetic and functional evidence from the scientific literature. We have completed filtration of 43 genomes. Altogether, 1387 variants in 670 genes remained for review after filtration. Approximately 15% of the variants were present in at least 5 individuals, and were therefore predicted to be clinically insignificant. To date, 25 individual genomes have undergone full interpretation. Of the 922 variants filtered from these 25 genomes, 48% were excluded primarily due to insufficient evidence for gene-disease associations and 11% were determined to be false positive either upon Sanger sequencing or based on analytical metrics. And, of the 922 variants, 15% were classified as Likely Benign/Benign, 18% as Uncertain Significance and 8% as Likely Pathogenic/Pathogenic, the later returned to patients at a rate of ~3 per patient. All variants and genes excluded from incidental findings return are made available for subsequent data annotation to prevent re-review. In summary, the creation and sharing of gene and variant exclusion datasets leads to a dramatic improvement in the efficiency of genomic variant interpretation. Although exclusion annotations are not currently submitted to ClinVar, we propose creating a mechanism for laboratories to share such datasets to aid in the efficiency of genomic data variant review.


14. Increased expression levels of FMR1 Iso10 and Iso20 mRNAs in premutation carriers Pretto DI. 1, Eid JS.2, Tang H-T.1, Loomis EW.1, Raske C.1, Durbin-Johnson B3, Hagerman PJ.1, Tassone F.1,4*

1Department of Biochemistry and Molecular Medicine, University of California, Davis, School of Medicine, Davis, California 95616, USA 2Pacific Biosciences, Inc., Menlo Park, California 94025, USA 3Department of Public Health Sciences University of California Davis, School of Medicine, Davis, California, USA 4MIND Institute, University of California Davis Medical Center, Sacramento, California 95817, USA.

Alternative splicing in the FMR1 gene generates proteins with likely different functional properties. Their function, cellular localization and/or levels of expression may be biologically important in particular in FMR1 associated disorders including Fragile X-associated tremor/ataxia syndrome (FXTAS).

A marked elevation in FMR1 mRNA transcript levels and reduced FMRP expression is observed in FMR1 premutation alleles (55-200 CGG repeats) although it is currently unknown if all or only some of the transcripts are overexpressed in premutations.

Thus, we have applied long-read sequencing by single molecule real time sequencing (SMRT®) to assess how many FMR1 sites of alternative splicing are used with a significant frequency and whether they are independent of each other.

We have determined which of the FMR1 mRNA transcripts are bonefide and more importantly which ones overexpressed in premutation carriers compared to controls. Our analysis, performed on lymphocytes, fibroblasts and brain samples derived from premutations and controls indeed revealed the existence of only 16 isoforms out of the 24 predicted variants in both control and premutation samples. Importantly, an altered distribution of isoforms spliced at both exons 12 and 14 was detected only in the premutations. Specifically Iso10 and Iso20, containing the complete exon 15 and differing only in splicing at exon 17 showed a 4-8 fold greater representation in all three tissues examined. Interestingly, the relative abundance of all other isoforms was similar between premutations and controls. qRT-PCR confirmed the presence of significant altered expression levels driven by isoforms simultaneously spliced at exons 12 and 14 in both lymphocytes (p < 0.001) and brain tissue (p=0.06) between premutations, with and without FXTAS, and age matched controls. Our results, in addition to show the feasibility of characterizing the isoform expression patterns in premutation and control samples by sequencing of full-length cDNA on a single-molecule level, underlying the importance of identifying the correct combination of different splice sites within full-length transcripts which if dysregulated can have a pathogenic effect.

These findings suggest that an unbalanced abundance of select isoforms may impose a biased function of FMRP in premutations. Further studies have yet to determine the impact of FMRP variants in the neurodevelopmental and neurological disorders associated with the FMR1 premutation.


15. ClinVar: Submitting Interpretations of Medical Variation

George R. Riley, Donna R. Maglott, Jennifer M. Lee, Wonhee Jang, Shanmuga Chitipiralla, Ricardo Villamarin-Salomon, Douglas Hoffman, Kenneth S. Katz, Michael Ovetsky, Amanjeev Sethi, Baoshan Gu, Wendy S. Rubinstein, Adriana Malheiro, Brandi Kattman, Melissa J. Landrum

National Center for Biotechnology Information (NCBI), National Library of Medicine, NIH, Bethesda, MD 20892

With the increasing use of genomic sequencing tests and accelerating pace of discovery of clinically significant variation, the need to aggregate variation data to support clinical interpretation is clear. ClinVar archives and versions submissions that include clinical significance of sequence variants with respect to a condition, along with supporting evidence. ClinVar reports conflicts of clinical significance from different submissions to enable users to gauge discrepancies and expert groups to submit their assertions on the data.

ClinVar, which launched its public webpage in April of 2013, currently has over 110,000 submissions from more than 125 submitters representing over 99,000 variants in more than 18,000 genes. Submitters include testing laboratories, the Breast Cancer Information Core (BIC) and other locus-specific databases, researchers, professional societies, as well as other resources such as OMIM® and GeneReviews®. ClinVar participates in the ClinGen collaboration, providing information for expert panel curation, and archiving the results of review.

ClinVar accepts submissions in several formats including spreadsheets, comma- or tab-separated text, and xml. Submitted variants are described in HGVS or genomic coordinate formats with a clinical interpretation in relation to a disease phenotype, along with optional additional information such as genotypes, observations in affected or unaffected individuals, animal models, in vitro assays, and in silico predictions. ClinVar provides significant quality assurance to submitters by: 1) verification of the reference sequence at the genomic location and the format of the variant, 2) reports to the submitter of internal inconsistencies in reported variant or clinical significance and 3) reports of clinical significance interpretation conflicts with other submissions. NCBI is developing an automated process to streamline submissions. ClinVar strongly encourages submitters to update their submissions with additional evidence, altered interpretations, and new variants.

ClinVar welcomes feedback and new submitters.


16. Identification And Characterization Of Marker Chromosome In a Patient with Turner’s Syndrome

Sonika Sharma, Priyanka Srivastava

Molecular Biology and Transplant Immunology Lab, Apollo Hospital, New Delhi, India

Introduction: We are describing a 14 year old female who was referred to us with severe growth retardation, primary amenorrhea, poor development of secondary sexual characteristics, web neck, increased FSH and LH. A provisional diagnosis of Turners syndrome was made clinically. On routine cytogenetic analysis she was found to be mosaic for 45,X and 46,X,+marker chromosome. The presence of a marker chromosome in Turner syndrome generally implicates a sex chromosome origin but it may also originate from a non-sex chromosome. If the marker chromosome originates from Y, the patient is at risk of developing Gonadoblastoma. Therefore, FISH test was performed for marker chromosome to rule out the presence of Y chromosome so as to delineate the risk of gonadoblastoma in the patient.

Methods: Peripheral blood sample was collected from the proband and cytogenetic examination was carried out using standard techniques of 72hr phytohemagglutinin stimulated peripheral blood lymphocyte culture. FISH was done using centromere enumerating probe for X and Y chromosome.

Results: The marker chromosome identified in the chromosomal analysis was found to be centromeric part of X chromosome on FISH analysis, thus ruling out the Y chromosome and the risk of gonadoblastoma.

Conclusion: We strongly recommend that FISH should be done in all cases of Turner syndrome where a marker chromosome is identified on chromosomal analysis so as to rule out the risk of gonadoblastoma and to provide appropriate genetic counseling.

Keywords: Turner’s Syndrome, Marker chromosome, Cytogenetics


17. A Male Infant with Partial Monosomy of 7p21 Syndrome Associated with Craniosynostois, Craniofacial Dysmorphism and Non Communicating Hydrocephalus T.D. Wanigasekera1, D. Sumathipala1, U.G.I.U. Kariyawasam1, A.V.L.K. Udalamaththa1, L.P.C. Saman Kumara2, R.W. Jayasekara1, V.H.W. Dissanayake1 1Human Genetics Unit, Faculty of Medicine, University of Colombo, Sri Lanka 2Castle Street Hospital for Women, Colombo, Sri Lanka A two weeks old male infant with dysmorphic features was referred to the Human Genetics Unit for genetic diagnosis. The clinical features of the child included craniosynostosis and facial dysmorphisms such as hypertelorism, bilateral epicathal folds with short upward slanting palpebral fissures, ptosis, flat nasal bridge, a long smooth philtrum, mild micrognathia and tented upper lip. He had prominent helical crura and transverse creases of hands bilaterally however there was no evidence of soft tissue syndactyly. Ultrasound scan of the brain revealed ventriculomegaly and 2D echocardiogram showed evidence of congenital heart disease: Patent Ductus Arteriosus and Atrial Septal Defect. An abdominal ultrasound scan was normal. There was no family history of similarly affected individuals. Karyotyping revealed a de novo terminal deletion of the short arm of chromosome 7 (46 XY,del(7)(p21pter). Among the developmental regulator genes found in this region TWIST 1 is the gene mostly matched with the infant phenotype. TWIST1 gene is located in 7p21 region and haploinsufficiency is associated with craniosynostosis. Micro deletion of chromosome 7p21.1 region is responsible for four types of syndromes: Craniosynostosis type1, Robinow Sorauf syndrome, Saethre-chotzen syndrome and Saethre-chotzen syndrome with eye lid anomalies. For further genotype phenotype correlation of this child targeted molecular genetic testing is required.

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