Arthur L. Beaudet - US grants

The objective of this project is to analyze gene regulation for the argininosuccinate synthetase (AS) locus. The intent is to determine the molecular basis for metabolite regulation by arginine and for enzyme overproduction in canavanine-resistant (Canr) cells. The mechanism of enzyme overproduction in Canr cells may be related to the mechanism of high organ specific expression in liver. Structural analysis of the AS gene and multiple pseudogenes is well advanced and will be completed. Attempts are in progress to obtain expression of AS using DNA mediated gene transfer with cDNA clones and minigene constructions. Canr variants of heterozygous citrullinemia cell lines will be used to distinguish whethe the Canr phenotype is cis- or trans-acting. The primary method for analysis of regulation will be the use of DNA mediated gene transfer attempting to demonstrate (1) metabolite regulation with transfected DNA and (2) altered regulation associated with the Canr phenotype either related to the recipient cells or to the transfected DNA. Functional regions of transfected DNA will be identified using deletion mutants and linker scanning mutants. Experiments designed to clone regulatory genes are also proposed. The proposed experiments are directly relevant to understanding regulation of gene expression in mammalian cells. This remains a high priority endeavor in biomedical research. Analysis of this human locus has direct relevance to the disease citrullinemia, and regulatory mutants may exist in addition to the well known structural mutants.

We have generated a human liver cDNA library and have developed a protocol for identifying cDNA clones within the library. The screening is based on pooling 5 to 10 cDNA clones on DBM filters and using plasmid selection of human or baboon poly(A)+RNA followed by immunoprecipitation and analysis on SDS polyacrylamide gels. The screening utilizes pooling of antibodies as well as cDNA clones so that a single lane in the gel analysis can represent 20 to 100 tests. We have identified a clone for human Alpha1-antitrysin and propose to use this clone to identify restriction fragment length polymorphisms to carry out prenatal diagnosis of the deficiency state. We propose to generate an improved, larger unfractionated cDNA library as well as 10 to 12 sublibraries based on fractionation of the poly(A)+RNA on a sucrose gradient. We propose to identify new cDNA clones for gene products of 0.1% or greater abundance. Currently screening is utilizing antibodies against argininosuccinate lyase, arginase, numerous apolipoproteins and numerous plasma proteins. We will focus specifically on attempts to clone cDNAs for carbamylphosphate synthetase, ornithine transcarbamylase and glucose-6-phosphatase. We will make the libraries available to other investigators. We will use DNAs for medical and biological applications such as molecular analysis of human diseases, prenatal diagnosis and gene mapping.

The overall goal of this project is to clone the gene for cystic fibrosis. If this can be accomplished, additional goals would be to explore the possibility of a heterozygote test suitable for use in the general population, and to explore any implications for therapy. A genomic DNA library has been prepared from a Chinese hamster cell hybrid which contains a small portion of human chromosome 7, highly likely to include the CF locus. Overlapping cosmids will be organized into contiguous DNA segments. Cosmids will also be grouped by mapping to pulsed field gel fragments. Genomic DNA regions from the cosmids will be evaluated using crossovers between linked markers and the CF locus, pulsed field gel electrophoresis and other strategies. Conserved and transcribed regions will be identified using Southern blotting with DNA from various species as well as Northern blotting analysis. Alternative strategies to clone the CF gene will be pursued using a concept of candidate gene families and strategies such as subtraction hybridization. For candidate genes identified using genomic DNA clones, cDNAs will be isolated and analyzed in attempts to prove whether they may or may not represent the CF gene product. CF cells will be analyzed by Southern blotting, by Northern blotting, by nuclease analysis of RNA and by nucleic acid sequencing to determine if a candidate gene is the CF gene. If the CF gene can be identified, efforts will be focused on development of a heterozygote test suitable for use in the general population. This could be based on a biological method or on a direct molecular analysis. In addition, longer term goals might focus on the potential for somatic gene replacement therapy using retroviral vectors encoding the CF gene product.

Numerous cell adhesion molecules found on leukocytes, endothelial cells, and platelets are involved in a central way in many biological functions and disease processes. These cell adhesion molecules include members of the immunoglobulin gene superfamily, integrins, selectins, and other proteins. This project focuses particularly on the beta-2 subunit of leukocyte integrins (CD18), intercellular adhesion molecule-1 (ICAM-1), and granule membrane protein-140 (GMP-140). The overall goals of this project are to generate mutations in the CD18, ICAM-1, and GMP-140 genes in the mouse using homologous recombination in embryonic stem (ES) cells. These mouse mutants would be used to study the role of these proteins in normal biological function and in disease. There is substantial evidence that these proteins play central roles in processes such as inflammation, autoimmune disease, infectious susceptibility, transplantation rejection, and perhaps atherosclerosis. The murine genes would be cloned and characterized in sufficient detail to prepare constructs for homologous recombination in ES cells. The CD18, ICAM-1, and GMP-140 genes would be interrupted by homologous recombination in ES cells using insertion and/or replacement vectors, tissue culture selection, screening with the polymerase chain reaction (PCR), and confirmation of homologous recombination with Southern blotting. Mutant ES cells would be introduced into mouse blastocysts to obtain chimeras and subsequently bred into the mouse germ-line. The phenotype of heterozygous and homozygous mutant mice will be examined clinically, pathologically, and physiologically. The effect of mutant phenotypes on murine autoimmune processes will be studied using model systems such as experimental allergic encephalomyelitis, lupus erythematosus, and diabetes mellitus. The effects of the mutant phenotypes on murine atherosclerosis will be examined using mice with putative atherosclerosis genotypes. Mutant mice will be made available on a collaborative basis and rapidly on an unlimited basis to other laboratories in order to facilitate analysis of other biological features of any mutant animals. There will be a sharp focus on obtaining null mutations in CD18, ICAM-1, and GMP-140 and on introducing these into the mouse germ-line in the first 1-3 years of the project.

DESCRIPTION: The objective of Core B1 is to provide developmentally based patient evaluations for persons enrolled in MRRC studies. These evaluations will be performed at the Meyer Center for Developmental Pediatrics, a unit operated collaboratively by Baylor College of Medicine and Texas Children's Hospital. This core will provide routine service in cases where billing is not feasible and more specialized evaluations are required for investigative purposes. The five major areas of investigation to which this core has contributed include studies of developmental outcomes following high-risk birth, the effects of CMV, measurement of mediation efficacy in treatment for AIDS, neurodevelopmental effects on dietary intake of polyunsaturated fatty acids, and clarification of the phenotypes of various genetic disorders. Eleven projects will use this core, including a number of studies of genetic syndromes, CMV, head injury, and AIDS. The cytogenetics core laboratory will perform classical cytogenetics on patients recruited in the research projects of MRRC investigators, and FSH mapping of genes or DNA markers. A total of 12 projects will require the services of this core facility. The Biochemical Genetics Laboratory provides testing for patients with inborn errors of metabolism. A small proportion of patients with mental retardation have single gene metabolic disorders that can be diagnosed by screening markers such as amino acids, organic acids, sugars, and mucopolysaccharides, etc. A second function of this core is to help investigators involved in mouse gene disruption by performing measurements of biochemical markers such as amino acids and organic acids as well as enzymatic assays. A third function is to perform follow up testing in patients with urea cycle disorders who would be receiving gene therapy. Hence, this is a metabolic markers screening and enzyme analysis laboratory which will be available for investigators of the MRCC.

The overall objective of this proposal is to develop improved methods for somatic gene therapy for treatment of lung disease in cystic fibrosis (CF). The rationale is that there is a great need for improved delivery systems which will combine high efficiency for transfection, delivery to the proper airway cells in the lung, and longer duration or permanent expression. All of the projects and almost all of the pilot projects are focused on this general objective. Project l has the following aims: reduction of toxicity of adenovirus (Ad) vectors using temperature sensitive and other mutations; development of Ad vectors based on alternative serotypes and nonhuman viruses; and development of Ad vectors for persistent expression. Project 2 has the following specific aims: development of Ad vectors accommodating much larger inserts (one use of which would be to include large segments of DNA to promote homologous recombination and permanent expression); and development of Ad vectors incorporating a regulatable promoter. Project 3 has the following specific aims: development of chemically defined delivery systems based on DNA conjugates using synthetic components to achieve receptor/Iigand binding, lysis of endosome, and nuclear targeting; and preparation of DNA conjugates for targeting to airway epithelium using ligands for the receptors for folate, mannose 6-phosphate, vasoactive intestinal peptide, and dimeric IgA. Project 4 has the following specific aims: comparison of aerosolization, tracheal instillation, and bronchial artery injection for delivery of Ad vectors to the airway epithelium; gathering .preclinical data for use of these routes of administration in humans; development of a program for clinical trials beginning with assessment of the natural history of Ad infections in CF patients and clinical electrophysiological measurements; assessment of bronchial artery and aerosol delivery of vectors developed in projects l and 3 and of AAV vectors from collaborators; and efficacy studies using CF mutant mice. Core A will prepare Ad vectors and retroviral vectors to meet FDA criteria for preclinical studies and human trials. Core B will provide an outstanding capacity for primate studies using new vectors and delivery systems from all four projects. Core C will provide histopathology, immunohistochemistry, and in situ hybridization studies. Topics of pilot projects include use of gene therapy for prevention of coronary restenosis, gene therapy for intestinal involvement in CF, development of additional mutations in the CF gene in the mouse, use of retroviral vectors for airway delivery, and development of a retroviral vector for nondividing cells.

We have shown that plasma concentrations of glutathione disulfide are increased in premature infants independent of respiratory system disease, suggesting that these infants are experiencing a significant oxidant stress even when exposed to low concentrations of supplemental oxygen. This oxidant stress correlates with a predisposition to develop "Oxygen radical mediated" chronic diseases, of which BPD is a prominent example. Animal models of oxygen radical mediated lung injury have largely centered around hyperoxia-exposure as the means of generating lung injury. We propose to generate adenoviral vectors which express a variety of antioxidant genes for administration to premature baboons which develop BPD in a classic model. 1-3 Initial studies will be performed using an adenovirus which bears the beta-galactosidase marker gene to determine the efficacy of adenoviruses as vectors for the delivery of transgenes to the lungs of ventilated prematures. Subsequent studies will examine the simultaneous delivery of adenoviral vectors and surfactant preparations to determine whether surfactant administration interferes with adenoviral transduction of pulmonary tissue. Successful transgene delivery in this setting would facilitate the treatment of RDS and BPD by permitting a dual approach to the prevention of lung injury. Initial studies of antioxidant gene delivery will be performed using a vector which contains the MnSOD gene. This will be delivered to premature baboons at 140 days gestation; they will be compared with untreated baboons using a clinical scale for severity of acute and chronic disease and by histologic comparison of lung tissue for the stigmata of BPD. Finally, a series of adenoviral vectors containing other antioxidant genes will be prepared; these will include glutathione reductase, glutathione peroxidase, gamma-glutamyl cysteine synthetase and nitric oxide synthase. These vectors will be used singly or in combination to discover the most effective treatment protocol for the prevention of oxygen toxicity in ventilated prematures.

This application is submitted by the organizers of the 1994 March of Dimes Birth Defects Foundation Clinical Genetics Conference entitled "Genes in Development and Cancer." The meeting is to be held at the Hyatt Orlando Hotel in Kissimmee, Florida on March 13-15, 1994. Funds are requested to defray the housing and travel costs of 20 graduate students, postdoctoral fellows, and junior investigators so they can attend this conference as active participants. Developmental aspects of the conference will include embryology, mouse/human homologies, Waardenburg syndrome, neural tube defects, neuronal migration defects, holoprosencephaly, limb abnormalities, hearing/deafness, and situs inversus. Cancer aspects of the conference will include Wilms tumor, Beckwith syndrome, p53 oncogene, cancer families, retinoblastoma, colon cancer, breast cancer, multiple endocrine neoplasia, neurofibromatosis, basal cell nevus syndrome, von Hippel-Lindau/renal carcinoma, DNA repair defects, and other topics. The conference will bring together investigators in a variety of basic and clinical sciences including molecular biology, cellular biology, embryology, developmental biology, cancer biology, clinical genetics, birth defects, and dysmorphology. The format includes 20-25 invited speakers, eight platform presentations selected from submitted abstracts, poster sessions, a round table on genetic counseling for cancer, and a session on contributed cases and diagnostic dilemmas. An audience of 300- 600 is anticipated with mechanisms to encourage extensive interaction among the attendees. There will be presentation of exciting new cell and molecular developments, but the planners of the conference have taken steps to assure the clinical relevance of presentations. Based on the previous experience of these annual meetings over the past 22 years, participation of young investigators in the 1994 conference will contribute significantly to the development of productive collaborations in the years to come. The three coorganizers with help from the other members of the Advisory Committee will evaluate written applications for travel support and will distribute funds on a competitive basis. The criteria for selecting travel award grantees include submission of an abstract of high quality original research, selection of the research for presentation, and financial need.

We have determined the in vivo flux through the urea cycle pathway by measuring the conversion of intravenously infused [15N-amide]glutamine to [15N]urea in both urea cycle patients and normal subjects. The patient population included neonatal onset citrullinemia and ornithine transcarbamylase deficiency (OTC) patients, symptomatic OTC heterozygotes, and asymptomatic OTC heterozygotes. Subjects were studied after maintenance on set protein and drug intake for 48 hrs. To evaluate the effects of protein intake, alternative route medications, and supplementary arginine or citrulline, patients and subjects were each studied twice. They were assigned to one of three groups:a) study on low and higher protein intake, b) study with and without Ucephan, or c) study with and without arginine. Enrichments of [15N] and 18O in plasma and urinary glutamine and urea were used to determine endogenous (urea cycle specific) and total urea synthesis. Neonatal onset patients exhibited zero [15N] enrichment consistent with an absence of endogenous urea synthesis, while OTC heterozygous females exhibited [15N] enrichment intermediate between normal controls and the citrullinemic patients. However, urea synthesis was markedly affected by either ucephan and arginine administration. We, therefore, derived an index of the proportion of endogenous to total urea synthesis as measured by the ratio of [15N]urea/[15N]glutamine. This index was found to be independent of diet, nutritional status, and alternative route medications. Moreover, the index correlated with severity of the clinical phenotype and exhibited little variability between serial studies. This index in combination with absolute measurements of urea synthetic rates will be useful for evaluating gene therapeutic interventions and for guiding the clinical management of urea cycle patients. These sutdies are now being extended to patients with other urea cycle defects.

DESCRIPTION (Adapted from the Investigator's Abstract): The overall goals of the project are (1) to understand the biology, genetics, and pathogenesis of diseases associated with deficiency of human chromosome 15q11-q13 and the homologous region of mouse chromosome 7, both of which contain numerous imprinted genes, and (2) to study selected basic aspects of imprinted gene expression. Paternal deficiency for human 15q11-q13 causes Prader-Willi syndrome (PWS) while maternal deficiency causes Angelman syndrome (AS). The first specific aim is to extend mutational analysis in AS patients in whom no molecular defect is identified to date with particularly emphasis on identifying additional missense mutations in UBE3A, the locus that is mutated in AS and encodes E6-AP ubiquitin protein ligase, and on identifying mutations in cis regulatory elements essential for expression of UBE3A. The second specific aim is to perform detailed phenotypic analysis of Ube3a knockout mice comparing maternal deficiency (AS) model, paternal deficiency, and homozygous deficiency with wild type mice. Emphasis will be on behavioral and learning studies, electrophysiological analysis of long-term potentiation, and immunohistochemical studies. The third specific aim is to perform an extensive genetic analysis of the PWS/AS region in the mouse by preparing a series of null mutations for Snrpn, Ipw, Gabrb3, Ndn, and newly identified genes. In addition, deletions will be prepared across intervals in the region with a focus on determining which loci account for the perinatal lethal phenotype associated with paternal deficiency for the region (potential equivalent to PWS) and on identifying cis regulatory elements upstream or downstream of Ube3A. The fourth specific aim is to define the putative imprinting center (IC) by constructing deletions upstream of Snrpn in the mouse to obtain mutations that prevent switching between paternal and maternal epigenotypes. The fifth specific aim is to develop a novel and broadly applicable method for visualizing regulation of gene expression through coat color variation and screen for cis- and trans- acting mutations that affect imprinted gene expression. Imprinted expression of the agouti cDNA under the control of the Snrpn promoter has been obtained, and ENU mutagenesis will be utilized. This project is directly relevant to the mental retardation found in PWS and AS, and the Ube3a mutant mouse has the potential to increase our understanding of learning and memory. The use of a coat color marker as a reporter for analysis of gene regulation in vivo in combination with mutagenesis has general applicability beyond imprinted gene expression.

The primary objective of the Mouse Genetics Core is to prepare various mutations in neuronal nicotinic acetylcholine receptors (neuronal nAChRs) and to provide these mice to the three projects and to the Morphology Core. The rationale for the overall strategy is based on the belief that genetic manipulation in the mouse is one of the most powerful methods available for delineating the function of genes and that this will be a valuable approach for understanding the role of nAChRs in nicotine addiction. Numerous mutations have already been transmitted to the mouse germline including alpha7 null, alpha7 L247T, alpha5 null, alpha3 null, beta4 null, and beta2 null. This core will provide the initial characterization of the phenotype for each mutation including survival analysis and basic clinical observations, Mice will be provided to the various collaborators for baseline neuropathological, behavior, and electrophysiological studies. When supplying mice to the various projects for in depth studies, the Core will provide genetic expertise and participate in experimental designs to determine breeding strategies, background genotype for mutations, types of mutations to be produced, feasibility of using blinded observations, numbers of animals needed for experimentation, and related aspects of each project. In addition to the mutations already available, the Core will produce the following mutations: alpha4 null, alpha3 conditional null, alpha7 conditional null, beta4 conditional null, and a null to L247T conditional mutation. The alpha4 null mutation will be prepared by deletion of essential exons. Conditional null mutations will be achieved by standard methods for preparation of flox premutations (exons flanked by loxP sites), and these will be bred to mice carrying inducible Cre transgenes or tissue-specific Cre transgenes to obtain inducible conditional mutations and tissue- specific conditional mutations as detailed in the proposal. Various double and triple mutations have been and will be produce using breeding of unlinked single mutations or introduction of multiple mutations within a single gene cluster in ES cells. In addition to providing the mutant mice to the projects within this proposal, mutant mice will be offered to the Jackson Laboratories for distribution within the research community shortly after publication. The mutant mice produced by this Mouse Genetics Core should prove invaluable for extensive studies involving the function of neuronal nAChRs an their role in nicotine addiction.

Prader Willi syndrome; diagnosis design /evaluation; orphan disease /drug; therapy design /development

The overall goals of this project are to further define the safety and toxicity of helper-dependent adenoviral (HD-Ad) vectors, to delineate the pathogenesis of the thrombocytopenia observed with the HD-Ad vectors, to attempt to identify mechanisms to circumvent the thrombocytopenia, to develop experience with these vectors in primates, and to move incrementally towards clinical trials with these very promising HD-Ad vectors. The pathogenesis of the thrombocytopenia will be studied in mice, focusing on direct interactions between platelets and vector and between endothelial cells and vector. One very important aim will be to evaluate safety, toxicity, and short-term expression using high doses of HD-Ad vectors in juvenile baboons. Because of the desire to initiate clinical trials with maximum safety, we are comparing the expression of factor IX and apolipoprotein A-I (apo A-I) with IM administration utilizing a muscle-specific promoter and IV administration aimed at expression in hepatocytes. Another aim will be to put in place all of the necessary reagents and commitments to prepare GMP quality HD-Ad vector suitable for use in pre-clinical and clinical studies. Toxicity studies with GMP quality vector will be conducted in mice and baboons. In longer-term experiments in baboons, we will test whether over-expression of apo A-I will protect against atherosclerosis in baboons. Finally, we propose to develop a clinical trial to introduce the HD-Ad vectors into the clinic using either IM or IV administration. No IRB-approved protocol is available at present, but the major possibilities under consideration include expression of factor IX in patients with hemophilia B or expression of apo A-I in patients with coronary artery disease and low production of apo A-I. The long-term significance of this project is to attempt to develop HD-Ad vectors designed to increase expression of the LDL receptor and/or apo A-I in humans. If successful, this approach could have a major impact on prevention or reduction of atherosclerosis in the human population.

The overall goals of this project are 1) to determine genotype/phenotype relationships across the chromosome 15q11-q13 imprinted domain and its mouse equivalent to include Angelman syndrome (AS), Prader-Willi syndrome (PWS), and other phenotypes; 2) to explore the molecular genetic and biochemical bases for regulation of genomic imprinting in mice and humans; 3) to expand the understanding of the role of genomic imprinting and epigenetics more generally in human disease; and 4) to explore therapeutic strategies for AS in mice and in cultured human cells as a model for epigenetic therapies. Initial gene trap experiments identified retinoblastoma binding protein 1 (Rbbp1) and Rbbp1-like-1 (Rbbp111) as having effects on imprinting; in addition, cultured cells lacking expression of the retinoblastoma (Rb) protein show altered DNA methylation and gene expression in both the Snrpn and H19 imprinted domains. The aims related to studies of regulation of genomic imprinting include the following: a) Continue genetic analysis of human chromosome 15q11-q13 and mouse 7C; b) Extend genetic screens in mice using ENU mutagenesis and perhaps sleeping beauty mutagenesis with new "yellow" Snrpn-agouti allele and in ES cells using gene-trap mutagenesis; c) Prepare null mutations in mice for Rbbp1 and Rbbp111 and characterize the phenotypes; d) Genetic analysis in mice using Mecp2, eed, Rbbp1, Rbbp111, Rb and other candidate genes and genes isolated in screens; and e) Biochemical and microscopy studies to include co-immunoprecipitation (IP) studies to identify protein-protein interactions, chromatin immunoprecipitation (CHIP) studies, immuno-histochemistry, immuno-FISH, and analysis of gene expression and DNA methylation. One goal is to validate whether five proteins identified by 2D gels of Purkinje cells from Ube3a mutant and wild-type mice are increased in abundance in vivo in mutant mice using immuno-histochemistry and western blots. Treatment for AS will be explored attempting to increase leaky expression of the silenced paternal allele of UBE3A. Studies will test the effect of various drugs on human cultured fibroblasts and in mice, and an ongoing clinical trial of folate and betaine will be modified based on these results. The hypothesis that intracytoplasmic sperm injection (ICSI) may cause imprinting defects will be tested in mice using a gene fusion method which allows detection of these defects based on coat color.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Rare cases of genetic or epigenetic diseases can provide major insights into more common factors of the same or similar phenotypes as exemplified by studies of homozygous familial hypercholesterolemia and more recently when a single deletion case led to the discovery of a gene causing CHARGE syndrome A few genetic or epigenetic conditions can present with typical autism including mutations in MECP2, mutations within chromosome 15q11-q13, rarely mutations in neuroligin genes, fragile X syndrome, and tuberous sclerosis. We hypothesize that more patients dianosed with autism spectrum disorders have mutations or epimutations involving MECP2, genes within chromosome 15q11-q13, and the loci causing fragile X syndrome and tuberous sclerosis than is currently recognized. We also hypothesize that genes that interact with MECP2 and UBE3A are candidate genes for mutation or epimutation causing autism. Careful studies of autism patients for changes in these loci have already led to additional insights into autism, especially for MECP2 and 15q11-q13. We propose to carry out in depth genotype/phenotype and epigenotype/phenotype correlations in autistic patients with known abnormalities in these genes and regions with the goal of achieving further insights into more common forms of autism. In addition, we will anlayze typical autism patients for novel forms of mutationor epimutation involving MECP2, genes within 15q11-q13, or other genes. We will perform mutation and epimutation alalyses for other autism candidate genes based on a) potential for explaining the male predominant sex ratio in autism as for brain- or synapse-related genes on the X or Y chromosome;or b) functional, biochemical, or regulatory relationships to MECP2, genes in the 15q11-q13 region, FRAXA, or the tuberous sclerosis genes. HYPOTHESIS 1. We hypothesize that more patients diagnosed with autism spectrum disorders have mutations or epimutations involving MECP2, genes within chromosome 15q11-q13, and the loci causing fragile X syndrome and tuberous sclerosis than is currently realized. 2. We hypothesize that more extensive genetic analysis and especially epigenetic analysis of these loci in autism patients will identify new causes of autism. 3. We hypothesize that patients with clinical diagnoses of Rett or Angelman syndrome without an identifiable molecular abnormality may have regulatory genetic or epigenetic defects involving the MECP2 or UBE3A, respectively. 4. We hypothesize that detailed phenotypic analysis of patients with known mutations or epimutations involving MECP2, chromosome 15q11-q13, and the other loci will allow selection and identification of additional autism patients with abnormalities of these loci. 5. We hypothesize that genes that interact with MECP2 and UBE3A are candidates for mutation or epimutation causing autism. 6. We hypothesize that brain- or synapse-related genes on the X or Y chromosome are candidates for mutations or epimutations causing autism.

DESCRIPTION (provided by applicant): High-density microarrays and next-generation sequencing technologies, coupled with the availability of the annotated human genomic sequence, are opening a road toward a comprehensive mapping of molecular epigenetic patterns. It is anticipated that comprehensive mapping and study of epigenetic patterns both horizontally along the genomic sequence and vertically across multiple differentiation and developmental stages and physiological conditions, will provide insights into human development, physiology, and disease. This vision of epigenomics calls both for novel organizational models suitable for high-throughput data-driven science and for innovative networked cyberinfrastructure. This project aims to develop such networked cyberinfrastructure and employ it to integrate and coordinate data analyses and data pipelines involving designated Reference Epigenome Mapping Centers (REMCs), NCBI, and other participants. The infrastructure builds on the now well established Genboree system which was developed in the context of numerous genome projects and has most recently been employed to coordinate the pilot stage of The Cancer Genome Atlas Project. The infrastructure will provide both scalability for further integration of new epigenomic technologies with increasing data production throughputs and adaptability to accommodate an increasing diversity of experimental and computational methodologies. Using the software-as-a-service model, web services, and semantic web technologies, the infrastructure will help integrate and coordinate efforts of REMCs, NCBI, and an increasing number of collaborating institutions and multi-disciplinary groups in the field of epigenomics working across geographic locations.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. ABSTRACT New evidence suggests that autistic disorder (AD) may be associated with abnormalities in folate metabolism, which is a process that affects genetic expression by facilitating the formation of methyl donors for DNA methylation. Limited data show that some children with AD show behavioral improvements with folic acid (FA) therapy, while others show a worsening effect. If behavioral worsening is linked with abnormalities in folate metabolism, then nutritional modifications could normalize these processes and result in clinical improvements. To address this premise, we propose a randomized, placebo-controlled crossover pilot study with two phases. The first phase will focus on the behavioral and biochemical responses of children with AD to high-dose folic acid supplementation. Because FA is an inactive folate that requires biochemical conversion to become active, and select genotypes impede this conversion, our general hypothesis is that FA will yield behavioral improvements in some children but exacerbate problem behaviors in others. During the second phase, children who had a worsened behavioral response to FA during phase 1 will participate in an open-label trial of high-dose Metafolin[unreadable] supplementation. The focus here would similarly be on the behavioral and biochemical outcomes of participating children following treatment with the study supplement. Because Metafolin[unreadable] is an active folate metabolite that should not be affected by genotypes in the folate pathway, our general hypothesis for phase 2 is that Metafolin[unreadable] would yield behavioral improvements without the risk for behavioral worsening. Results from this project may provide support for continued study of the potential relationship between folate metabolism and problem behaviors among children with AD, potentially justifying the need to examine effects of folate supplementation among a larger sample of affected children.

Human disease conditions are often very complex due to disturbed interdependencies inside the human body rather than to the impairment of a single cell function. Cell culture models are therefore not sufficient as a model system: full information about human disease conditions can only be obtained from the analysis of a whole animal organism. This is particulariy important in complex systems where cultured cells are not able to model physiological processes. The laboratory mouse is the closest organism to humans that can be easily studied by genetic manipulation. The ultimate goal of many IDDRC projects is to understand the genetic basis of abnormal brain development and/or mental retardation/development disabilities in humans. To achieve this goal many IDDRC investigators, require the generation of genetically modified mice with specific mutations in the germ line and somatic tissues to study the roles of genes In vivo. The core will allow investigators to generate special alleles permitting conditional deletions and conditional rescues studies. Such conditional alleles are not currently available through any other resource. Given that several IDD have already exhibited the potential of reversibility with re-establishment of gene expression, the ability to create mouse models that permit testing for recovery is critical as groups pursue pathogenesis and interventional studies. In addition to conditional studies, targeting of non-coding miRNAs is in demand to gain insight into post transcriptional regulation of various IDD genes. Thus this core augments the large scale efforts to provide knockout alleles for every mouse gene. The procedures necessary for the manipulation of ES cells and the creation of GEM are capital intensive and technically challenging. Dedicated and experienced staff and costly equipment are required to produce mutants in timely cost-effective manner. The ES Core makes it possible for IDDRC investigators to quickly and efficiently obtain mouse models for research relating to developmental disabilities. The Core also provides an invaluable service to many non-IDDRC investigators throughout Baylor and throughout the United States.

DESCRIPTION (provided by applicant): Project Summary/Abstract. The long-term objectives of this proposal are to define genotype/phenotype correlations for the extended Prader-Willi/Angelman domain on chromosome 15q11-q13 that stretches from Breakpoint 1 (BP1) to Breakpoint 5 (BP5), with special emphasis on the BP4-BP5 region where deletions are associated with mental retardation, autism, epilepsy, schizophrenia, and bipolar disorder. Genetic analysis will focus both on copy number variations (CNVs) deleting or duplicating multiple contiguous genes and on point mutations within individual genes, especially CHRNA7. Previous work on this project has focused on Prader- Willi syndrome (PWS) and Angelman syndrome (AS), caused by paternal and maternal deficiency, respectively, for the central portion of the domain. The focus is now being broadened to include autism caused by duplications of this region and to study phenotypes associated with the flanking portions of the domain, BP1 to BP2 and BP4 to BP5. Deletions of chromosome 15q13.3 (BP4-BP5) remove six contiguous genes, and a smaller deletion removes CHRNA7 and one exon of an adjacent gene. Aim 1 is to determine genotype/phenotype relationships for the BP4-BP5 region with emphasis on the CHRNA7 gene. Aim 1a is to determine if the 15q13.3 and CHRNA7 duplications are pathological or benign using case control studies. Aim 1b is to identify the basis for the phenotypic heterogeneity associated with the 15q13.3 and CHRNA7 deletions focusing primarily but not exclusively on various genetic modifier effects. Aim 1c is to search for loss-of- function point mutations in CHRNA7 in particularly high risk samples such NIMH samples with both epilepsy and schizophrenia or both epilepsy and bipolar disorder. Aim 2 is to determine genotype/phenotype relationships for the BP1-BP2 region and the four genes therein. Aim 3 is to determine the molecular basis for the parent of origin effects of duplications of the BP1/BP2 to BP3 region which typically cause autism when on a maternal chromosome and are usually benign when on the paternal chromosome. This aim will emphasize expression analysis (increasingly relying on RNA-Seq) and epigenetic studies of human brain tissue comparing duplications of the 15q11-q13 region with controls. Aim 4 is to determine the function of the snoRNA HBII-85 cluster, because there is now relatively strong evidence that paternal deficiency for this snoRNA cluster causes the major components of the PWS phenotype. This aim will focus on genome-wide analysis of expression and on alternative splicing and RNA editing (again using RNA-Seq) in human and mouse brain lacking expression of HBII-85. A series of target candidate genes have been identified by others based on bioinformatic analysis, and further bioinformatic analysis is planned. The expression of these candidate genes will be studied in control and PWS brain from mouse and human using RT-PCR to analyze alternative splicing and RNA editing. PUBLIC HEALTH RELEVANCE: Project Narrative. This project has strong medical relevance, first because genetic variation in this chromosomal region and especially in one gene (CHRNA7) is common and definitely can cause mental retardation, autism, epilepsy, schizophrenia, and bipolar disorder. The second strong medical relevance is to autism because duplications of this region on the maternal chromosome cause autism, while duplications on the paternal chromosome do not, and understanding of the molecular basis for this difference should shed considerable light on the causes of autism that are not caused by changes in DNA sequence. Third, snoRNAs are a little studied form on noncoding RNAs, and understanding their function is of importance, because deficiency of one class of snoRNAs is a major component of the cause of Prader-Willi syndrome and likely is relevant to obesity.

DESCRIPTION (provided by applicant): Annotation of the reference human genome has identified approximately 20,000 protein coding genes as well as 3,000 non-coding RNAs. Together these genes orchestrate the development of the organism, supporting all aspects of the function of cells, tissues, organ systems as well physiology and behavior. The l000 genomes project has revealed extraordinary levels of diversity in human genomes, yet for most genes neither the function of normal version nor the disease consequence of loss-of-function variants is known. The mouse provides a route to understand the function of genes and their variants. Mice share developmental, physiological, anatomical and metabolic parallels with humans, which are evident in healthy as well as diseased states. These reflect similarities of the genes in both species. Mutant mice generated using ES cell technology are a sensitive biological assay system from which a deep understanding of function can be gleaned and they also provide a long lasting biological resource for further study. The objective of this proposal is to generate mutant mice from a resource of ES cells with conditionally targeted, lacZ-tagged alleles generated under previous NIH (KOMP) and EU (EUCOMM) funded programs. We and others will use these mice to discover the function of genes. We are proposing to carry out this work at scale and have formed a consortium of three Institutes (Baylor College of Medicine, the Sanger Institute and MRC Harwell) to work together as equal partners to achieve this objective. We will generate mice corresponding to 1500 mutant genes from ES cells distributed by the Sanger Institute. We will characterize the adult and embryonic expression pattern of the each targeted gene and by breeding determine the requirement of each for embryonic development and fertility, if any. All of the alleles will be cryo-preserved and placed in a repository and all of the data will be deposited in a centralized data coordination center to support further studies. RELEVANCE: Most of the 20,000 genes in a typical person are normal but we also have several hundred broken ones. While some broken genes can cause severe disease such as cystic fibrosis or cancer, others have little or no consequence, or function only under stress. Currently we have some understanding of the function of just one third of human genes. If we are to fully understand human health and disease we must expand knowledge of gene function to all of our genes.

DESCRIPTION (provided by applicant): A comprehensive functional annotation of all genes is a key goal for the future investigation of mammalian systems and biomedical sciences. We have established a consortium for the large-scale phenotyping of mouse mutants, which is fundamental to the investigation of gene function. The BaSH consortium, Baylor College of Medicine (BCM), Houston, Texas, the Wellcome Trust Sanger Institute Mouse Genetics Programme, Hinxton, United Kingdom, and the Medical Research Council Harwell, (Mammalian Genetics Unit and Mary Lyon Centre), United Kingdom, will undertake broad-based phenotype analysis of 300 IKMC mouse lines per year with the aim of identifying perturbations on developmental, physiological and biochemical pathways that will guide experimenters to develop hypothesis-driven research into disease systems. Our aims are to 1) complete the broad-based disease phenotyping of over 1500 lines of mutant mice in the C57BL/6N genetic background, 2) validate an optimized and enhanced broad-based phenotyping pipeline that will detect a variety of disease phenotypes and increase throughput, and 3) submit phenotypic data to the designated data coordination center, ensuring an interface with the wider biomedical scientific community that will inform human genetic studies. Our approach is to build on our unique expertise in mouse phenotyping and the successful operation of major pilot projects for mouse phenotyping of EUCOMM and KOMP mutants to deliver a phenotyping pipeline with strategic breadth that serves the needs of the medical community. Our pipeline design aims to deliver mouse models in key therapeutically relevant areas - for example in Cardiovascular, Metabolic, Neurological, Respiratory and Immunological Systems. Assessment of mouse mutants using our phenotyping pipeline will discover novel preclinical models of therapeutic importance, encompassing many of the diseases that account for the highest rates of disease morbidity throughout the developed world. RELEVANCE: Most of the genes in a person are normal, but we also carry several hundred broken ones. While some broken genes can cause severe disease such as cystic fibrosis or cancer, others have little of no consequence, or function only under stress. Currently, we have some understanding of the function of just one third of human genes. If we are to fully understand human health and disease we must expand knowledge of gene function to all of our genes using model organisms such as the mouse.

Adolescent; Adult; Age; Alleles; Angelman Syndrome; Animals; Antisense Oligonucleotide Therapy; Antisense Oligonucleotides; Antisense RNA; base; Binding; Biological Markers; Birth; Body Fluids; Brain; brain morphology; Cell Culture Techniques; Cell model; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Data; design; Development; developmental disease/disorder; Diagnosis; Dose; drug development; Evaluation; Fibroblasts; Funding; Gene Activation; Genes; Genetic Transcription; Genomic DNA; Genotype; Goals; Government; Histopathology; Human; imprint; improved; induced pluripotent stem cell; Intellectual functioning disability; knock-down; Life; Measures; Mediating; Mental Retardation and Developmental Disabilities Research Centers; metabolomics; Methods; Molecular Genetics; mouse model; Mus; Mutation; Neuraxis; Neurodevelopmental Disorder; Neurologic Examination; Neurons; Oligonucleotides; optimism; Patients; Persons; Pharmaceutical Preparations; Pharmacologic Substance; Phase I Clinical Trials; Phenotype; Plasma; preclinical study; Proteomics; Provider; Research; Route; Schedule; Sequence Homology; Skin; Spinal Muscular Atrophy; Staging; Technology; Testing; Therapeutic Index; Therapeutic Intervention; therapy development; Time; Tissues; transcriptomics; Wild Type Mouse;

Annotation of the reference human genome has identified approximately 20,000 protein coding genes as well as 3,000 non-coding RNAs. Together these genes orchestrate the development of the organism, supporting all aspects of the function of cells, tissues, organ systems as well physiology and behavior. The lOOOgenome project has revealed extraordinary levels of diversity in human genomes, yet for most genes neither the function of normal version nor the disease consequence of loss-of-function variants is known. The mouse provides a route to understand the function of genes and their variants. IVIice share developmental, physiological, anatomical and metabolic parallels with humans, which are evident in healthy as well as diseased states. These reflect similarities of the genes in both species. Mutant mice generated using ES cell technology are a sensitive biological assay system from which a deep understanding of function can be gleaned and they also provide a long lasting biological resource for further study. The objective of this proposal is to generate mutant mice from a resource of ES cells with conditionally targeted, lacZ-tagged alleles generated under previous NIH (KOMP) and EU (EUCOMM) funded programs. We and others will use these mice to discover the function of genes. We are proposing to carry out this wor(^ at scale and have formed a consortium of three Institutes (Baylor College of Medicine, the Sanger Institute and MRC Harwell) to work together as equal partners to achieve this objective. We will generate mice corresponding to 1500 mutant genes from ES cells distributed by the Sanger Institute. We will characterize the adult and embryonic expression pattern of the each targeted gene and by breeding determine the requirement of each for embryonic development and fertility, if any. All of the alleles will be cryo-preserved and placed in a repository and all of the data will be deposited in a centralized data coordination center to support further studies. RELEVANCE (See instructions): Most of the 20,000 genes in a typical person are normal but we also have several hundred broken ones. While some broken genes can cause severe disease such as cystic fibrosis or cancer, others have little or no consequence, or function only under stress. Currently we have some understanding of the function of just one third of human genes. If we are to fully understand human health and disease we must expand knowledge of gene function to all of our genes.

Consortium for large-scale production and phenotyping of knockout mice (UM1) ABSTRACT: The long-term goal of the International Knockout Mouse Consortium (IKMC) is to develop a resource of targeted mutations in mice for every protein-coding gene in the mammalian genome that the research community can use to elucidate gene function in human biology and disease. We have formed a consortium of two Institutes (Baylor College of Medicine and MRC Harwell). This application describes our plan to generate null alleles for 1500 mouse genes using CRISPR/RGN genome editing technology and validate each line using established QC procedures. We will cryopreserve all mutant strains and deliver germplasm to the MMRRC repositories. These mutant alleles represent a gold standard resource of mutant alleles for the wider community, and an important foundation for future research and translational studies using the mouse models created. We will perform broad-based adult phenotyping on all mutant lines, and for the first time incorporate an ageing component for a significant fraction of mutants, the latter involving an additional testing component from 12 months onwards. We will also assess homozygous lethal and subviable lines in an embryonic phenotyping pipeline, It is apparent that there is a relationship of mouse lethal (essential) and subviable genes with human disease loci. All allele and phenotype data will be submitted in real time to the Data Coordination Center, ensuring that all of the BasH data is disseminated to the wider biomedical scientific community. We will continue our R&D program to introduce appropriate methodological and technological developments from production to phenotyping. BasH will continue to pilot improvements in Cas9 RGN for the production of more sophisticated alleles, and in parallel ensure that developments in cryopreservation approaches at Baylor and Harwell are integrated into the production pipeline. We will also continue our major projects in improving and enriching the adult and embryo phenotyping pipelines, focusing on areas such as metabolomics, behavioral phenotyping and home cage monitoring, with the aim of providing more complex and longitudinal data. Baylor College of Medicine and MRC Harwell have the established expertise, experience, and resources to efficiently and cost-effectively meet this goal.

Consortium for large-scale production and phenotyping of knockout mice (UM1) ABSTRACT: The long-term goal of the International Knockout Mouse Consortium (IKMC) is to develop a resource of targeted mutations in mice for every protein-coding gene in the mammalian genome that the research community can use to elucidate gene function in human biology and disease. We have formed a consortium of two Institutes (Baylor College of Medicine and MRC Harwell). This application describes our plan to generate null alleles for 1500 mouse genes using CRISPR/RGN genome editing technology and validate each line using established QC procedures. We will cryopreserve all mutant strains and deliver germplasm to the MMRRC repositories. These mutant alleles represent a gold standard resource of mutant alleles for the wider community, and an important foundation for future research and translational studies using the mouse models created. We will perform broad-based adult phenotyping on all mutant lines, and for the first time incorporate an ageing component for a significant fraction of mutants, the latter involving an additional testing component from 12 months onwards. We will also assess homozygous lethal and subviable lines in an embryonic phenotyping pipeline, It is apparent that there is a relationship of mouse lethal (essential) and subviable genes with human disease loci. All allele and phenotype data will be submitted in real time to the Data Coordination Center, ensuring that all of the BasH data is disseminated to the wider biomedical scientific community. We will continue our R&D program to introduce appropriate methodological and technological developments from production to phenotyping. BasH will continue to pilot improvements in Cas9 RGN for the production of more sophisticated alleles, and in parallel ensure that developments in cryopreservation approaches at Baylor and Harwell are integrated into the production pipeline. We will also continue our major projects in improving and enriching the adult and embryo phenotyping pipelines, focusing on areas such as metabolomics, behavioral phenotyping and home cage monitoring, with the aim of providing more complex and longitudinal data. Baylor College of Medicine and MRC Harwell have the established expertise, experience, and resources to efficiently and cost-effectively meet this goal.

Consortium for large-scale production and phenotyping of knockout mice (UM1) ABSTRACT: The long-term goal of the International Knockout Mouse Consortium (IKMC) is to develop a resource of targeted mutations in mice for every protein-coding gene in the mammalian genome that the research community can use to elucidate gene function in human biology and disease. We have formed a consortium of two Institutes (Baylor College of Medicine and MRC Harwell). This application describes our plan to generate null alleles for 1500 mouse genes using CRISPR/RGN genome editing technology and validate each line using established QC procedures. We will cryopreserve all mutant strains and deliver germplasm to the MMRRC repositories. These mutant alleles represent a gold standard resource of mutant alleles for the wider community, and an important foundation for future research and translational studies using the mouse models created. We will perform broad-based adult phenotyping on all mutant lines, and for the first time incorporate an ageing component for a significant fraction of mutants, the latter involving an additional testing component from 12 months onwards. We will also assess homozygous lethal and subviable lines in an embryonic phenotyping pipeline, It is apparent that there is a relationship of mouse lethal (essential) and subviable genes with human disease loci. All allele and phenotype data will be submitted in real time to the Data Coordination Center, ensuring that all of the BasH data is disseminated to the wider biomedical scientific community. We will continue our R&D program to introduce appropriate methodological and technological developments from production to phenotyping. BasH will continue to pilot improvements in Cas9 RGN for the production of more sophisticated alleles, and in parallel ensure that developments in cryopreservation approaches at Baylor and Harwell are integrated into the production pipeline. We will also continue our major projects in improving and enriching the adult and embryo phenotyping pipelines, focusing on areas such as metabolomics, behavioral phenotyping and home cage monitoring, with the aim of providing more complex and longitudinal data. Baylor College of Medicine and MRC Harwell have the established expertise, experience, and resources to efficiently and cost-effectively meet this goal.

Consortium for large-scale production and phenotyping of knockout mice (UM1) ABSTRACT: The long-term goal of the International Knockout Mouse Consortium (IKMC) is to develop a resource of targeted mutations in mice for every protein-coding gene in the mammalian genome that the research community can use to elucidate gene function in human biology and disease. We have formed a consortium of two Institutes (Baylor College of Medicine and MRC Harwell). This application describes our plan to generate null alleles for 1000 mouse genes using CRISPR/RGN genome editing technology and validate each line using established QC procedures. We will cryopreserve all mutant strains and deliver germplasm to the MMRRC repositories. These mutant alleles represent a gold standard resource of mutant alleles for the wider community, and an important foundation for future research and translational studies using the mouse models created. We will perform broad-based adult phenotyping on all mutant lines, and for the first time incorporate an ageing component for a significant fraction of mutants, the latter involving an additional testing component from 12 months onwards. We will also assess homozygous lethal and subviable lines in an embryonic phenotyping pipeline, It is apparent that there is a relationship of mouse lethal (essential) and subviable genes with human disease loci. All allele and phenotype data will be submitted in real time to the Data Coordination Center, ensuring that all of the BasH data is disseminated to the wider biomedical scientific community. We will continue our R&D program to introduce appropriate methodological and technological developments from production to phenotyping. BasH will continue to pilot improvements in Cas9 RGN for the production of more sophisticated alleles, and in parallel ensure that developments in cryopreservation approaches at Baylor and Harwell are integrated into the production pipeline. We will also continue our major projects in improving and enriching the adult and embryo phenotyping pipelines, focusing on areas such as metabolomics, behavioral phenotyping and home cage monitoring, with the aim of providing more complex and longitudinal data. Baylor College of Medicine and MRC Harwell have the established expertise, experience, and resources to efficiently and cost-effectively meet this goal.