UW CENTER FOR MENDELIAN GENOMICS
Ashok Jay
University Of Washingtoncity: Seattle country: United States (us)
Grant 1U54HG006493-01 from National Human Genome Research Institute
Abstract: Over the past three decades, the genes underlying nearly 3,000 Mendelian disorders have been identified by methods such as linkage analysis and positional cloning. Although the availability of a reference human genome greatly accelerated these efforts, there are thousands of additional suspected Mendelian disorders that remain unsolved. An understanding of the genetic basis of a Mendelian disorder can yield fundamental insights into basic human biology and disease pathophysiology, as well as a molecular basis for diagnosis or carrier status determination. In some instances, biological insights from studying Mendelian disorders can prove highly relevant to our understanding of more common diseases. Recently, we and others have shown that the coupling of targeted capture and next-generation DNA sequencing technology can be used to cost-effectively determine nearly all coding variation in an individual human genome, a process termed exome sequencing. We, and others, have also demonstrated how exome sequencing can be applied to efficiently identify the causal genes for Mendelian disorders that have proven intractable to conventional modes of analysis. To accelerate progress towards a comprehensive understanding of the genetic basis of all Mendelian disorders, we propose to establish the UW Center for Mendelian Genomics. Our proposal has four specific aims (1) To organize samples for all unsolved Mendelian disorders from investigators around the world, either by their submission to our center for sequencing, or by their inclusion on a public sample list that we will develop; (2) To apply our existing production pipeline for exome and genome sequencing to samples corresponding to unsolved Mendelian disorders, and to improve this process through ongoing technology innovation; (3) To determine the genetic basis for as many unsolved Mendelian disorders as possible, through efficient study design and effective, innovative analysis; (4) To take a leadership role in the dissemination of methods and data
Keywords: Affect; analytical method; base; biobank; Biological; Candidate Disease Gene; Chromosome Mapping; Clinical; Code; Collaborations; Communities; Complex; cost; Coupling; Data; database of Genotypes and Phenotypes; Databases; Deposition; Development; Diagnosis; Disease; disease-causing mutation; DNA; DNA Sequence; Employee Strikes; Ensure; Equilibrium; Exhibits; exome; Family; Functional disorder; Gene Mutation; Genes; Genetic; genetic linkage analysis; Genome; genome sequencing; genome-wide; Genomics; Goals; Hereditary Disease; Human Biology; human disease; Human Genome; Human Genome Project; improved; Individual; Inheritance Patterns; innovation; insight; Investigation; Leadership; Link; Medical Genetics; Methods; Molecular; new technology; next generation; Online Systems; Open Reading Frames; Pathway Analysis; positional cloning; Process; Production; programs; Protocols documentation; public health relevance; Rare Diseases; Reporting; repository; Research; Research Design; Research Personnel; Resources; Role; Sampling; Sequence Analysis; sharing data; success; Technology; Universities; Variant; Washington; Work
Relevance: Mendelian disorders are rare diseases caused by mutations in single genes. Over the past three decades, the genes underlying thousands of Mendelian disorders have been identified. However, there are thousands of additional Mendelian disorders, the genetic basis for which has yet to be determined. We propose to apply new technologies and new analytical paradigms to efficiently determine the genetic basis of nearly all unsolved Mendelian disorders
Project start date: 2011-12-05
Project end date: 2015-11-30
Budget start date: 5-DEC-2011
Budget end date: 30-NOV-2012
1U54HG006493-01 (2012): $1950000
Sponsored Links Excellgen http://Excellgen.com
Grants awarded to Ashok Jay
ULTRASENSITIVE IDENTIFICATION AND PRECISE QUANTITATION OF LOW FREQUENCY SOMATIC M
Ashok Jay, Assistant Professor
University Of Washingtoncity: Seattle country: United States (us)
Grant 1R21CA160080-01 from National Cancer Institute
Abstract: The ultrasensitive detection of clinically relevant somatic alterations in cancer genomes has great potential for impacting patient care, e.g. for early detection, establishing diagnoses, refining prognoses, guiding treatment, and monitoring recurrence. However, current technologies are poorly suited to the robust detection of somatic mutations present at very low frequencies. Massively parallel sequencing represents one path forward, but its sensitivity to detect very rare events is fundamentally constrained by the sequencing error rate. Our goal is to develop a new experimental paradigm that overcomes this limitation. In our approach, each copy of a target sequence that is present in a sample is molecularly tagged during the first cycle of a multiplex capture reaction with a unique barcode sequence. After amplification, target amplicons and their corresponding barcodes are subjected to massively parallel sequencing. During analysis, the barcodes are used to associate sequence reads sharing a common origin. Through oversampling, barcode-associated reads error-correct one another to yield an independent haploid consensus for each progenitor molecule, i.e. "molecular counting". Furthermore, the collapsing of commonly derived reads inherently corrects for any allele-specific bias during amplification, such that estimates of mutant allele frequency can be accompanied by precise confidence bounds. In our first aim, we will develop experimental methods and analytical tools that enable the robust detection of targeted somatic mutations via molecular counting to frequencies as low as 1 mutated copy in a background of 100,000 unmutated copies. In our second aim, we will develop three ultrasensitive, multiplex molecular counting assays that are specifically targeted at panels of clinically relevant cancer mutations or genes, and rigorously evaluate these for reproducibility. The availability of robust, cost-effective, generically applicable tools for the ultrasensitive, multiplex detection of rare somatic events will be a transformative step forward for the translation of discoveries in cancer genetics to a clinical setting. As we enter an era of "personalized medicine", DNA sequencing technology will be increasingly important to public health, contributing towards the unraveling of the genetic basis of human disease, as well as for clinical diagnostics. This proposal aims to develop ultrasensitive methods for detecting cancer-relevant mutations in tumor samples. These technologies have the potential to directly enable the translation of discoveries made in cancer genetics to clinical applications such as the early detection of cancer and the monitoring of patients for cancer recurrence
Keywords: Algorithms; Alleles; analytical tool; base; Biological Assay; cancer genetics; cancer genome; cancer genomics; cancer recurrence; Cell Line; Clinical; clinical application; clinically relevant; Code; Consensus; Consensus Sequence; cost effective; cost effectiveness; Detection; Development; Diagnosis; Diagnostic; digital; DNA; DNA Sequence; Early Diagnosis; Event; Frequencies (time pattern); Gene Frequency; Genes; Genetic; Genetic Heterogeneity; genome sequencing; Genotype; Goals; Haploidy; human disease; Malignant Neoplasms; Medicine; Methods; Molecular; Monitor; Monitoring for Recurrence; multiplex detection; mutant; Mutate; Mutation; Mutation Detection; Nucleotides; Oncogenes; outcome forecast; Patient Care; Patient Monitoring; progenitor; Protocols documentation; public health medicine (field); Reaction; Reading; Reagent; Reproducibility; research study; Sampling; Screening for cancer; Secondary to; single molecule; Somatic Mutation; Surveys; Technology; tool; Translations; tumor; Tumor Suppressor Genes
Relevance: As we enter an era of "personalized medicine", DNA sequencing technology will be increasingly important to public health, contributing towards the unraveling of the genetic basis of human disease, as well as for clinical diagnostics. This proposal aims to develop ultrasensitive methods for detecting cancer-relevant mutations in tumor samples. These technologies have the potential to directly enable the translation of discoveries made in cancer genetics to clinical applications such as the early detection of cancer and the monitoring of patients for cancer recurrence
Project start date: 2011-09-16
Project end date: 2014-08-31
Budget start date: 16-SEP-2011
Budget end date: 31-AUG-2012
PFA/PA: RFA-CA-10-005
1R21CA160080-01 (2011): $227632
IDENTIFICATION OF CANINE MINOR HISTOCOMPATIBILITY ANTIGENS
Ashok Jay, Assistant Professor
Fred Hutchinson Cancer Research Centercity: Seattle country: United States (us)
Abstract: PROJECT 2 IDENTIFICATION OFCANINE MINOR HISTOCOMPATIBILITY ANTIGENS Project 1 has developed an approach at DLA-identical canine hematopoietic cell transplantation (HCT) that results in stable mixed donor-host chimerism. Persistent host hematopoiesis can serve as an experimental model of persistent hematologic malignancy seen in some patients transplanted under Projects 3 and 4. Conversion of mixed to all-donor chimerism can be achieved with injection of donor lymphocytes that have been sensitized to host minor histocompatibility antigens expressed on peripheral blood mononuclear cells (PBMC), however at the price of often fatal graft-vs.-host disease (GVHD). T-cell responses directed against ubiquitously expressed minor antigens are thought to be responsible for GVHD, while T-cell responses against a combination of ubiquitous and hematopoietic-specific minor antigens contribute to elimination of residual host hematopoietic cells in a manner analogous to the graft-vs.-leukemia effect observed in human patients. The identification of minor antigens restricted to hematopoietic cells therefore holds great promise for improving allogeneic HCT outcomes. That knowledge would facilitate the development of sensitization strategies that target host hematopoietic cells while sparing GVHD target tissues. However, while the dog model of allogeneic HCT is optimal for preclinical development of novel HCT therapies, no canine minor, histocompatibility antigens have been described to date, and existing methodologies for minor antigen identification are inefficient. To address this problem, we propose a novel approach to minor antigen discovery in the dog. This approach utilizes next generation sequencing technology to define protein coding variations unique to the recipient and expressed in PBMC which will be used for sensitizing the HCT donor. Sensitized donor T-cells will then be injected into the respective recipients with the aim of converting mixed to full-donor chimerism and causing GVHD. After conversion has been accomplished, T-cells will be harvested from recipients and tested for responses against candidate minor antigens using a novel, high- throughput T-cell assay. Positive responses would define genuine minor histocompatibility antigens. Using qRT-PCR, we will then identify those minor antigens that are highly expressed in hematopoietic cells but not in GVHD target tissues. Next, relevant minor antigen peptides will be used to sensitize donor T-cells with the aim of converting mixed to full-donor chimerism without GVHD (Project 1). Eventually, this concept will be tested in a canine model of acute leukemia in Project 1. Benefits to Public Health Taken together, the studies proposed in this Project and the in vivo studies proposed in Project 1 have the potential of developing new and effective approaches benefiting patients with persisting/relapsing malignancies treated by allogeneic HCT under Projects 3 and 4
Keywords: Acute leukemia; Address; Age; Alleles; allelic variant; Allelomorphs; Allogenic; Allografting; Animals; Antigens; Assay; ATGN; base; Bioassay; Biologic Assays; Biological Assay; Blood (Leukemia); Body Tissues; Breeding; Cancers; canine; Canine Species; Canis familiaris; Cell Transplantation; Cells; Chimerism; Ciclosporin; Clinic; Code; Coding System; Collaborations; Comorbidity; conditioning; cost; CsA; Custom; Cyclosporin A; Cyclosporine; Cyclosporine A; Cyclosporines; Cyclosporins; Data; design; designing; Development; Disease; disease/disorder; Disorder; Dogs; domestic dog; Donor Lymphocyte Infusion; drug/agent; Drugs; experience; Experimental Models; Experimental Models, Other; Functional RNA; gene product; Gene variant; Genes; Genetic; Genetic Diversity; Genetic Polymorphism; Genetic Variation; Genomics; Genotype; Goals; Graft-Versus-Host Disease; Graft-vs-Host Disease; GVHD; Harvest; Hematologic Cancer; Hematologic Malignancies; Hematologic Neoplasms; Hematological Malignancies; Hematological Neoplasms; Hematological Tumor; Hematopoiesis; Hematopoietic; Hematopoietic Cancer; Hematopoietic Cellular Control Mechanisms; high risk; High Throughput Assay; high throughput screening; Histocompatibility Antigens; Homologous Wasting Disease; Human; Human, General; Immunization; immunogen; Immunologic Stimulation; Immunological Stimulation; Immunostimulation; Immunosuppressants; immunosuppression; Immunosuppression Effect; Immunosuppressions (Physiology); immunosuppressive; Immunosuppressive Agents; Immunosuppressive Effect; improved; in vivo; Injection of therapeutic agent; Injections; Knowledge; language translation; leukemia; Leukemias, General; lymph cell; Lymphocyte; Lymphocytic; malignancy; Malignant Hematologic Neoplasm; Malignant Neoplasms; Malignant Tumor; Mammals, Dogs; Man (Taxonomy); Man, Modern; Medication; Method LOINC Axis 6; Methodology; Minor; Minor Histocompatibility Antigens; Minor Histocompatibility Peptides; Minority; MMF; Modeling; Models, Experimental; Molecular; Mother Cells; mycophenolate mofetil; Mycophenolate Mofetil (Cellcept); mycophenolic acid morpholinoethyl ester; Natural immunosuppression; neoplasm/cancer; neoral; new approaches; next generation; Non-Coding; Non-Coding RNA; novel; novel approaches; novel strategies; novel strategy; Outcome; Patients; PBMC; Peptides; peripheral blood; Peripheral Blood Mononuclear Cell; Pharmaceutic Preparations; Pharmaceutical Preparations; polymorphism; Polymorphism (Genetics); Polymorphism, Genetic; Polymorphism, Single Base; pre-clinical; preclinical; prevent; preventing; Price; pricing; Principal Investigator; Procedures; Progenitor Cells; programs; Programs (PT); Programs [Publication Type]; Proteins; Public Health; public health medicine (field); pup; Reaction; Regimen; Relapse; Residual; Residual state; response; reverse transcriptase PCR; Reverse Transcriptase Polymerase Chain Reaction; Risk; Role; RT-PCR; RTPCR; Runt Disease; Sandimmun; sandimmune; SangCya; Sensitization, Immunologic; Sensitization, Immunological; Shotgun Sequencing; Single Nucleotide Polymorphism; SNP; SNPs; social role; Stem cells; T cell response; T-Cells; T-Lymphocyte; Technology; Testing; thymus derived lymphocyte; Thymus-Dependent Lymphocytes; Tissues; Translating; Translatings; transplant; transplant patient; Transplant Recipients; Transplantation; Transplantation Antigens; tumor; Variant; Variation; Variation (Genetics)
Budget start date: 1-FEB-2011
Budget end date: 31-JAN-2012
5P01CA078902-13_8823 (2011): $332203