Howard J. Jacob - US grants

Despite intensive investigation, the etiology of insulin dependent diabetes mellitus (IDDM) remains unclear. Studies in the human, mouse, and rat indicate that a gene or genes in the major histocompatibility complex (MHC) explain some--but not all of the inherited predisposition to the disease. The other genetic factors remain obscure. We propose to use the Diabetes Prone (DP) BB rat, which among the best models of human IDDM with onset and pathogenesis closely resembling the human disease. Using several crosses involving the DP rat, we have been able to map two genes (Iddm1, Iddm2) responsible for IDDM, and have evidence that there is one additional gene (Iddm3) that appears to confer resistance in the Fischer rat. Specifically, we propose to: 1) Map the gene responsible for conferring diabetes resistance in the Fisher Rat. By genotyping backcross progeny with markers from our genetic linkage map of the rat, we will be able to scan the entire genome, simultaneously and identify regions of the genome important in the development of diabetes. 2) Use positional clone techniques to identify Iddm1 and Iddm3. This strategy will require the construction of contigs spanning the gene containing regions. A yeast artificial chromosome (YAC) library will be constructed for the rat and used to make these contigs. Once the genes have been cloned, we will characterize the function of these genes. 3.) Study the role of the MHC in the development of diabetes. The MHC (Iddm2) is well known to play a major role in IDDM, yet the mechanism remains obscure. To study the role of the MHC, we will introgress the MHC from the Fischer and Lewis rats on to a DP background. Genetic markers flanking the MHC will be used to track the introgression. These congenic lines will be used to determine the role of different MHC classes in an otherwise diabetes permissive background. 4) Analysis of Iddm1 and Iddm3 Homologues in Human Pedigrees. Human homologues of Iddm1, Iddm3 and any other non-MHC found to co-segregate with IDDM in the DP rat, will be developed into genetic markers. In collaboration with Ake Lernmark's lab, we will assay these markers in human pedigrees using affected sib-pair analysis and population association studies. These studies should identify genetic loci responsible for diabetes in both the BB rat and man.

Vertebrate embryologists and developmental biologists are becoming increasingly interested in a new model system, the zebrafish, Brachydanio rerio. The interest in zebrafish is derived from its value as both a genetic and embryological organism. It is ideal for genetics because the animals are small, hardy and easy to raise in large numbers because: the females lay 100-200 eggs each spawning, which can occur 1-2 times per week, and techniques for mutagenesis and transgenesis have been well- defined. For embryological studies, their advantage is that fertilization is external, and the embryos are nearly transparent, so that the migration of cells and the earliest stages of organogenesis can be directly observed. Many mutant fish have been isolated after using chemical, radiation, or insertional mutagenesis, and at least three mutagenesis screens are currently underway with the goal of reaching saturation within the next few years. The zebrafish is the only vertebrate with these advantages. Specifically we propose to: l) Construct the first complete genetic linkage map of the zebrafish. Our goal is to have a map with at least a 10 cM resolution. 2) Develop a reference cross (1000 zebrafish), and map this cross. This reference cross is essential for initiating fine structure mapping, and facilitating positional cloning. The reference cross will be made available to the entire zebrafish community. 3) Establish a means of resource distribution, insuring marker information is accessible to investigators, and that DNA from the reference cross is easily obtainable. What would the map provide and is this the time to invest in building a genetic linkage map in zebrafish? A map would 1) allow synteny conservation mapping; 2) allow complete integration of a single map, and reference cross for mutations discovered by the zebrafish community; and 3) build the foundation for gene identification and positional cloning. The zebrafish field is rapidly expanding, and there are hundreds of investigators now working on zebrafish and specific zebrafish mutations. If map construction is delayed, not only will funds be expended by several laboratories to make rudimentary independent maps of specific regions, but funds will also have to be allocated to integrate the various "local" maps. For these reasons, and after discussion, the largest zebrafish facilities decided to focus on construction of genetic linkage map using simple sequence length polymorphisms (SSLPs) at the MGH. It is for this reason funds are requested.

Hypertension is an inherited polygenic disease, and its expression modified by the environment. Molecular genetic techniques provide the opportunity for dissecting the genes responsible hypertension, but only one locus has been shown, definitively to cosegregate with high blood pressure humans. Major limitations in human studies include: l) small numbers of ascertained patients, 2) etiological heterogeneity (different genes in different patients), 3) technical and theoretical issues concerning a total genome scan using affected sibpairs. Consequently, genetic dissection in human has been limited to candidate genes. In contrast, the rat genetic models of hypertension are more tractable for genetic dissection and several loci which cosegregate with high blood pressure identified. Unfortunately, these studies used small crosses, focused on one locus at a time and did not control for genetic background effects. We propose to use four large crosses, controlled for genetic background effects, to determine genetically "hot" loci to be studied in our patient populations. This approach reduces the number of genotypes to be done by our Human Genotyping Center and provides candidate regions, demonstrated to play a role in genetic susceptibility to hypertension. Identification of the "hypertensive" genes is likely to involve positional cloning and physiological characterization, neither of which are likely to be accomplished in human. To facilitate the detection of genetic "hot" spots and to facilitate the rapid transition from rat to human, we propose to: 1. Locate genetically "hypertensive" loci using several large rat crosses. The best candidate genes are likely to be those identified in several crosses. Furthermore minimizing genetic background affects should increase our chance of identifying loci that act as modifiers and control expression of hypertension in humans. This strategy is analogous to that accomplished for type 1 diabetes [1]. 2. Convert regions of interest in the rat to human homologs. Conservation of gene order and use of radiation hybrids will facilitate the quick transition to genetic markers for genotyping in humans. 3. Map candidate genes in the human and in the rat. Only a limited number of candidate genes have been mapped in the rat, human or both. The localization of a candidate gene serves as a starting point, and if correct will obviate the need for positional cloning. 4. Identify the genes responsible for hypertension. Another goal of this RFA is to identify these genes. Our genetic network are focused on the genetic localization, we will however initiate the identification by constructing congenic lines of rats. Congenic rats will facilitate postional cloning, physiological characterization and studies on environmental interaction.

Why some patients with hypertension develop end stage renal disease (ESRD) and other patients with similar levels of blood pressure do not, is poorly understood. In addition, the number of patients with hypertension-associated ESRD continues to increase despite improved antihypertensive management and early detection programs. We have been studying the relationship between hypertension and ESRD using the genetically hypertensive fawn-hooded rat model (FHH/EUR). The FHH is characterized by the early presence of systemic hypertension, glomerular hypertension, progressive proteinuria and focal glomerulosclerosis, resulting in premature death due to renal failure. Treating FHH rats with an ACE inhibitor, reduces blood pressure and proteinuria, pointing to an important role for blood pressure in the pathogenesis of renal failure in this model. The interaction between hypertension and renal failure is studied using two approaches: classic physiological characterization and molecular genetics. A gene accounting for 26% of the genetic variance in systolic blood pressure was mapped to chromosome 1. This gene appears to have minimal impact on proteinuria and therefore, renal failure. However, we did locate two other genes on chromosome 1, renal failure 1 (Rf-1), and renal failure 2 (Rf-2) that together are responsible for 46% of the genetic variance in proteinuria. Importantly, Rf-l failed to account for any of the variance in blood pressure--demonstrating a genetic predisposition to renal failure in the FHH. Accordingly, we propose three specific aims: 1. Identify the Human Homologues of the Rat Rf-1 and Rf-2 genes. Providing the IRPG1 group the opportunity to determine whether the rat homologues play a role in human ESRD. 2. Clone and Identify Rf-1. Since ESRD is a multifactorial disease (polygenic with environmental interaction), the probability of cloning the gene responsible for ESRD initially in humans is remote. We will construct a congenic rat that canoes only Rf-1 thereby, reducing the model to a single gene enabling us to use standard well-studied methodologies for positional cloning. 3. Study the Interaction between Blood Pressure and the Rf-1 Gene. Continued physiological characterization of this model is essential now that we understand that the FHH has a genetic predisposition for renal failure. With the discovery of Rf-1, we will have the opportunity to study the effect of blood pressure on renal failure in our FHH and congenic models. These studies provide our best hopes of understanding the etiology of hypertension-associated ESRD.

DESCRIPTION (Adapted from applicant's abstract): The rat remains a major model for biomedical research within both academia and industry. The strength of the rat is the long history of physiological, pharmacological, and biochemical studies that characterizes this model system. However, until recently the rat has not been a major "genetic system." With the success of the Human Genome Project in mapping and identifying genes responsible for monogenic diseases, there has been a shift towards the multifactorial disorders. The rat offers a distinct advantage in that there are more than 140 inbred strains of rats, most of which are model systems for these multifactorial disorders (1). The broad utility of the rat is perhaps best illustrated by the large number of NIH Institutes, Centers and Office of the Director that provide the support for the rat genome project and this RFA. Generation of a cDNA or expressed sequence tag sites (ESTs) project will further accelerate the development of the rat as a "genetic system". In order to make the ESTs useful to the whole research community, the ESTs need to be arrayed, sequenced and attached to the rat gene map. With a large number of genes and ESTs mapped a conserved gene order map is derived linking the rat genome to the genomes of the human and the mouse, in which the sequence and therefore all the genes are known. Using the principles of conserved gene order between mammalian species, investigators can use this gene order as the first approximation of the gene order in the rat. For example, there are 181 conserved linkage groups between mouse and human (2). I. COMPLETE THE FRAMEWORK MAP OF THE RAT RADIATION HYBRID (RH) PANEL AND MAP A SUBSET OF ESTs. In order for the rat radiation hybrid (RH) panel to be useful as a general mapping tool, a framework map is required. The investigators will begin by II. TO ARRAY AND SEQUENCE A KIDNEY EST LIBRARY. In year 1, while the other Centers that are sequencing ESTs are coming on-line, the investigators will complete their kidney EST sequencing project using the rat cDNA library from Dr. M. Bento Soares.

Why some patients with hypertension develop end stage renal disease (ESRD) and other patients with similar levels of blood pressure do not, is poorly understood. In addition, the number of patients with hypertension-associated ESRD continues to increase despite improved antihypertensive management and early detection programs. We have been studying the relationship between hypertension and ESRD using the genetically hypertensive fawn-hooded rat model (FHH/EUR). The FHH is characterized by the early presence of systemic hypertension, glomerular hypertension, progressive proteinuria and focal glomerulosclerosis, resulting in premature death due to renal failure. Treating FHH rats with an ACE inhibitor, reduces blood pressure and proteinuria, pointing to an important role for blood pressure in the pathogenesis of renal failure in this model. The interaction between hypertension and renal failure is studied using two approaches: classic physiological characterization and molecular genetics. A gene accounting for 26% of the genetic variance in systolic blood pressure was mapped to chromosome 1. This gene appears to have minimal impact on proteinuria and therefore, renal failure. However, we did locate two other genes on chromosome 1, renal failure 1 (Rf-1), and renal failure 2 (Rf-2) that together are responsible for 46% of the genetic variance in proteinuria. Importantly, Rf-l failed to account for any of the variance in blood pressure--demonstrating a genetic predisposition to renal failure in the FHH. Accordingly, we propose three specific aims: 1. Identify the Human Homologues of the Rat Rf-1 and Rf-2 genes. Providing the IRPG1 group the opportunity to determine whether the rat homologues play a role in human ESRD. 2. Clone and Identify Rf-1. Since ESRD is a multifactorial disease (polygenic with environmental interaction), the probability of cloning the gene responsible for ESRD initially in humans is remote. We will construct a congenic rat that canoes only Rf-1 thereby, reducing the model to a single gene enabling us to use standard well-studied methodologies for positional cloning. 3. Study the Interaction between Blood Pressure and the Rf-1 Gene. Continued physiological characterization of this model is essential now that we understand that the FHH has a genetic predisposition for renal failure. With the discovery of Rf-1, we will have the opportunity to study the effect of blood pressure on renal failure in our FHH and congenic models. These studies provide our best hopes of understanding the etiology of hypertension-associated ESRD.

The long-term objectives of this project are to identify novel genetic loci contributing to familial frequency of end-stage renal disease in humans. The specific aims of this protocol are to confirm data previously given by probands with ESRD via a questionnaire, to collect further data about their medical history and family structure and, to collect blood samples of affected and other family members.

DESCRIPTION (provided by applicant): The Rat Genome Database provides a core resource for rat researchers combining genetic, genomic, pathway, phenotype and strain information with a focus on disease. The goal of RGD is to provide investigators with a research platform that facilitates the elucidation of disease mechanisms by implementing standard data formats and ontologies. To meet this goal, we propose three specific aims: 1) To acquire, integrate and functionally annotate emerging genomic elements and variations along with core data to create a comprehensive genome resource. New gene models, sequence and map data and variations such as single nucleotide polymorphisms (SNPs), copy number variants (CNVs), and splice variants will be integrated through collaborations with NCBI and Ensembl and the use of innovative data pipelines. Curators will continue to focus on functional annotation of core data using multiple ontologies. New information including chemical-gene, drug-gene interactions and their impact on biology or disease will be added. Tools will be developed for mining, analysis and visualization of new data types. Educational activities for this aim will focus on new users and new tools for existing users. 2) To create a comprehensive phenome resource including phenotype measurements and strain medical records. We will develop a phenome database and provide individual strain medical records to provide easy access to the richness of this information. The phenome resource will include specific educational activities focused on phenotyping protocols, breeding and the use of our new tools and strain resources. 3) To link genotypes (Aim 1) to phenotypes (Aim 2) through QTLs, molecular, cellular and physiological pathways and the disease portals. RGD will continue to curate QTL data and enhance the QTL reports to provide a navigational hub linking genotype and phenotype data. Drug and physiological pathways will be curated in addition to disease related signaling and regulatory pathways and interactive diagrams will be used to link pathways, biological processes, genomic variations and phenotype data. RGD will expand its Disease Portals to serve as integration points for genomic and phenotype data, disease model profiles, and pathway data. Educational activities will focus on tools for comparative studies between rat and human, as well as those which integrate genotype and phenotype data.

The NHLBI Family Blood Pressure Program is made up of four cooperating networks whose overall objective is to localize and characterize genes contributing to variation in blood pressure levels and hypertension status. The four networks were originally separately funded and competitive, but two critical realizations have led to full cooperation and collaboration. First, the oligogenic nature of blood pressure control dictates that large samples are necessary to achieve adequate statistical power for genomic linkage and association analyses. Second, linkage intervals are broad and contain large numbers of genes, so that success in identifying genes and mutations requires the effort of multiple laboratories freely sharing information. This coordination extends far beyond phenotyping and genotyping and is best exemplified by the Program's creation of a pooled data set and agreements about coordinated publications. During the initial funding period, the Program surpassed its original recruitment goals, carried out multiple genome-wide linkage and association analyses and created an interim pooled data set consisting of phenotype and genotype data from more than 10,000 individuals. In this renewal application, the Program proposes five specific aims to be carried out by all four networks. These aims can be grouped according to two complementary themes: First, these applicants will create and analyze a database of blood pressure- related phenotype and genotype data from all FBPP participants (Aim 1). Within linked regions, they will identify allelic variation within positional candidate genes and evaluate the relationship of these polymorphisms with blood pressure levels and hypertension status (Aims 2 and 3). Second, they will use quantitative measures of target organ damage to identify genes that influence susceptibility to develop hypertensive heart and kidney diseases (Aims 4 and 5). In addition to the Program specific aims, each network proposes specific aims to be carried out by that network alone, based on unique aspects of their population and interests and expertise of the investigators. The Family Blood Pressure Program represents the most determined multidisciplinary approach to the genetics of hypertension ever assembled. The resulting synthesis of ideas and amassed data permits rigorous hypothesis testing not otherwise possible and will hasten understanding of the previously elusive genetic variation responsible for disease risk.

The present PGA proposes a novel and powerful approach to dissect multigenic traits through the development of panels of chromosomal substitution strains of rats (consomic rat panels). In a panel of consomic rats, a single chromosome is replaced one at a time, so that the contribution of genes on each chromosome can be assessed by phenotyping the consomic strain for the traits of interest. Consomic rat panels enable one to assess the contribution of genes specific to that chromosome by reducing the number of QTL differences in each test and providing inbred strains with a uniform genetic background. The present PGA is therefore designed to develop, phenotype, and distribute 88 consomic strains in the form of reciprocal chromosomal substitutions. The program is built upon the existing strengths in physiology, genomics, and bioinformatics which exist within the institution. These strengths will enable the investigators to link biological functions of heart, lung, and blood systems to genomic data, development a renewable national resource for investigators to study the impact of multiple disease genes on systems biology, and provide basic information which investigators can use to understand the impact of allelic variance and their interactions with the environment upon diseases that influence the heart, lung, and blood systems. Environmental stressors such as hypoxia, exercise, and high salt intake will be used to unmask deficiencies in normal homeostatic mechanisms and idiopathic mechanisms that contribute to disease. Towards this goal, the investigators are proposing to develop and distribute 88 strains of rats in the form of reciprocal chromosomal substitution strains (consomic) of rats in which the investigators have used 317 phenotypes to characterize heart, lung, kidney, vasculature, and blood function in response to environmental stressors (hypoxia, exercise, salt intake). The investigators will utilize comparative mapping strategies to link these traits to the genomes of the mouse and human. This PGA will build upon the existing infrastructure and programmatic experience in physiological genomics and bioinformatics, to increase an understanding of the genetic basis of fundamental mechanistic pathways of the heart, lung, kidney, blood and vasculature responses to stress. The dissemination of these data and resources to a large number of investigators will provide a valuable new tool to allow the translation of genes to function and disease.

The Genomics Core, under the direction of Dr. Jacob, will serve Projects by Cowley, Roman, and Greene in the genotyping[unreadable] of the congenic animals, sequencing, and providing bioinformatics support for all the Projects and Cores. This[unreadable] Core generates all wet lab genomic data related to genotyping the rats to generate the congenic and[unreadable] subcongenic animals using simple sequence length polymorphisms (SSLPs), generation of SSLP markers,[unreadable] cloning, sequencing, single nucleotide polymorphism (SNP) detection and physical mapping (if needed). Dr.[unreadable] Jacob has extensive experience in all facets of this Core through his last 16 years of research. Dr. Jacob[unreadable] directs the Human and Molecular Genetics Center (HMGC) at the Medical College of Wisconsin (MCW). This[unreadable] center has in place all the personnel, equipment, robotics and laboratory information management systems[unreadable] required to meet the needs of this PPG. The genotyping component, highly automated, is set up to make early[unreadable] breeding decisions, which allows us to minimize the cost of rat housing. The Genomics Core will also direct[unreadable] many of the activities related to mutation detection and gene identification in Projects by Roman and Greene. Finally, gene[unreadable] hunting, high through-put sequencing and comparative genomics all require significant bioinformatics[unreadable] infrastructure. Dr. Jacob is the PI for the Rat Genome Database (RGD) and directs the Bioinformatics[unreadable] Program at MCW. As a result the analytical tools necessary for data analysis, gene identification and[unreadable] prediction, and gene expression analysis are all in place and operational.

ESRD is a major health problem in the United States, with increasing incidence. Hypertension and diabetes account for more than 67% of ESRD. These associations are not understood, but many believe there is a final common pathway for renal failure independent of inducing agent or disease. Not everyone with hypertension or diabetes develops ESRD, and we demonstrated that renal failure genes could be independent of hypertension. We have a comprehensive research program utilizing genetics, genomics, transgenics to study the interactions of hypertension, diabetes and renal disease susceptibility genes. The goal of this proposal is to identify genes responsible for susceptibility to hypertension-related ESRD, and to identify the physiological pathways involved. We propose three specific aims: 1. Validate that Rab38 is the Rf-2 gene. We recently reported Rab38 to be the Rf-2 gene. We hypothesize that the Rab38 gene has pleiotropic effects and contributes to the development of proteinuria in FHH rats. We will test this hypothesis by a) studying albumin uptake in primary cultures of proximal tubule and b) with transgenic "rescue". 2. Complete the positional cloning and validation of the Rf-1 gene and Rf-4 gene. The Rf-1 QTL isthe dominant locus for ESRD in the FHH. Importantly, this locus has a strong interaction with Rf-4. We have reduced both the Rf-1 interval to 2.5 cM and the Rf-4 interval to 0.8cM, and the entire region has been sequenced in BN rats to a nearly finished level. We will investigate each locus using sub-congenics, physiological studies and sequence comparison to identify the causal gene(s) in both loci. Causal genes will be validated by rat transgenic rescue in the FHH genome background. 3. Initiate the positional cloning of Rf-3. We have demonstrated our ability to clone genes by position. We show that Rf-3 is also in a region linked to renal disease in mouse and human as is the case for Rf-1,-2, and -4. We will use the existing double (Rf-1+3) congenic strain to generate the overlapping congenics in the Rf-3 interval. We are confident that we will either find the gene or narrow down the interval to a handful of genes when we complete the term of this grant.

DESCRIPTION (provided by applicant): End-stage renal disease (ESRD) remains a major health problem in the United States, with an incidence that has been increasing steadily for more than a decade. More than 67% of ESRD is associated with hypertension and/or diabetes. The causes for these associations are not known. However, there is increasing evidence that susceptibility genes are likely to play a major role in determining a patient?s predisposition to ESRD. We have developed a comprehensive research program including genetics, genomics, rat transgenics and mechanism-based physiology to study the complex interaction between hypertension and susceptibility genes for renal disease. Here we propose to focus these tools on the following specific aims: 1. Complete the positional cloning of the Rf-1 gene. We identified this important QTL and propose to combine mapping and comparative genomics to locate the specific gene. 2. Determine pathways involved in the renal disease process using microarray technology. By identifying genes differentially expressed in normal and diseased kidney over the time course of ESRD development, we will see how gene expression is modified in the disease process. 3. Test our hypothesis that Rf-1 causes impaired renal autoregulation due to a lack of myogenic tone, resulting in glomerular hypertension and renal damage. We will also pursue mechanism-based studies of the QTL?s Rf-2, -3 and -5. 4. Study the Rf-1 locus by constructing transgenic rats carrying YACs. This will allow a direct test of any genes within the YAC. Transgenics will also be used to validate Rf-1. 5. Use comparative genomics to study human homologue of the Rf-1 region, Rf-1 gene and other candidate genes in humans. Using affected/unaffected human sibpairs, we will use SNP and sequence analysis to determine if individuals exhibiting ESRD show a significant association with sequence variants in candidate genes.

This PGA proposes to continue the success of the PhysGen program by capitalizing on a variety of technological advances, extending the data by manipulating specific genes and assessing the results physiologically in three different genetic backgrounds. The "functionality" of relevant genes will be tested by generating knockouts (KO) in rats, using a novel strategy that combines ENU mutagenesis with a yeast truncation assay. This technology will enable identification of animals with KOs of targeted genes of interest before weaning. The genes selected for KO have been derived from those that have been linked to heart, lung and blood disorders using molecular genetic, genomic and expression technologies during the first four years of all 11 PGA programs. In our preliminary studies, one of these genes (Avpla) was targeted and knocked out. As genomic background is shown to play a major role in the expression of phenotypes in KO mice, this PGA will attempt to make the KOs in 3 genetic backgrounds in the rat, the SS, BN, and FHH strains. The selection of these 3 strains will also enable PhysGen to leverage the enormous amount of phenotypic data generated on these strains during the first four years, notably 8,610 physiological data points per strain, including baseline and stressors, providing an unprecedented level of baseline data for a mutagenesis screen. Many traits have been demonstrated to be phenotypic extremes in two off these three strains. For example, hypertension is in the FHH and SS, but not the BN; whereas pulmonary traits! are abnormal in the BN and FHH, but normal in the SS. Knocking genes out in a disease background, as well as on a "normal" model is a departure from the traditional strategy for making new disease models. Presumably removing a key gene in a disease pathway will ameliorate the trait in some cases, and exacerbate the phenotype in other cases. Testing in animals with a disease amounts to a sensitized screen, where we are looking to "prevent" expression of the disease phenotype, as has been done in lower organisms. Once KOs are made on the inbred strains, multiple KO models can be rapidly made, as well as crossing the KOs into the 44 consomic strains to assess the impact of both a gene KO and a different physiological background. There are five specific aims to achieve the overall goals of this program: 1) Attempt to KO 70 genes that have been studied by the PGA that can be used in conjunction with our consomic strains, 2) characterize the KO rats physiologically, 3) participate in trans-PGA activities, 4) develop new data analysis tools for managing disparate data types, and for linking this information to the mouse and human data generated, and 5) provide an education component.

DESCRIPTION (provided by applicant): One of the key limitations to understanding the genetic basis of complex human disease is the availability of good models. The laboratory rat is one such model which has tremendous value because of intensely studied physiological, pathological and sequence similarities to humans. More than 1300 disease-related quantitative trait loci (QTL) have been mapped in genetic studies in the rat. There is a disconnection, however, in the rat research field between physiology and genetics because there are no efficient means to mutate and identify genes. We have been developing a new tool to close this technology gap. We are now building upon preliminary data showing that transposon insertional mutagenesis using the Sleeping Beauty (SB) transposon system can generate heritable mutations in the rat at a rate which is 50-fold more efficient than conventional methods. We believe that properties of SB transposition, specifically the tendency for transposons to jump to nearby sequences within a chromosome (local hopping), will allow us to target these regions for mutagenesis and dramatically accelerate disease gene discovery. Another powerful application of transposon mutagenesis is cancer gene discovery. In somatic cells of transgenic rodents, SB transposon insertional mutagenesis can drive tumor formation by the rare, but repeated mutation of cancer genes. We can use the transposon to find these cancer genes by studying their insertion sites in tumor DNA. We specifically propose to develop these two applications in parallel to model genetic disease by 1) developing multiple transgenic strains which have transposons in different regions of the genome to take advantage of local hopping for transposon mutgenesis within QTL regions and 2) develop the SB transposon system for somatic mutagenesis in the rat. We chose the Dahl salt-sensitive (SS) inbred rat strain as our model because it is at the forefront of cardiovascular research among many researchers and we have preliminarily characterized it as a new model for human breast cancer. We will combine the SS strain and SB transposon mutagenesis with decades of physiological and pathological characterization to create the disease models and discover the disease genes which are involved. Narrative: The key for researchers to develop diagnostic testing and treatments for complex human diseases like cardiovascular disease and cancer is finding the genes that are responsible. We use what we call `genetic model organisms'like rats to understand the pathology of different diseases and work hard to discover genes that are involved by mutating, or removing them, and determining what happens. We are developing a powerful new tool which is much more efficient at doing this in the rat so that we can accelerate research in many disease areas to improve the quality of life for all of us.

DESCRIPTION (provided by applicant): Complex human diseases, such as hypertension and renal disease, are major health problems in the United States. The National Heart, Lung, and Blood Institute (NHLBI) has invested in many genome-wide association studies (GWAS) and other types of genetic and genomic studies to provide an understanding of the molecular pathophysiological mechanisms underlying complex human traits and diseases. Although many genes and regions have been associated with hypertension, the roles of many of these genes in the underlying mechanisms have not been rigorously tested. Animal models provide the ability to dissect the complex interactions between multiple risk factors and environmental factors. This proposal will combine a powerful, new methodology for site directed mutagenesis in the rat with our experience in physiological studies investigating vascular and renal mechanisms controlling blood pressure. The novel technology for gene knock-outs (KO) in the rat will allow us to knock-out a large number of genes nominated by the GWAS and combine these gene knock-outs with hypertensive, genetically susceptible and normotensive rat strains. Specifically, we propose the following aims. Aim 1 - Investigate the mechanistic relationships between the genes and known mechanisms of hypertension and renal disease. A two tier system will be used to investigate the mechanisms involved in long-term maintenance of blood pressure and hypertension. Level one investigates blood pressure, baroreceptor reflex, oxidative stress, vascular reactivity, and response to salt load in 20 KO strains. Level two will study therapeutic pharmacogenetics, pressure-natriuretic-diuretic relationships and renal hemodynamics, and vascular mechanisms. Aim 2 - Knock-out 100 genes in a sensitized strain. The genes to be targeted will be selected by a committee using criteria focusing on replication in human genetic studies, lack of knowledge of the gene and its pathway, comparative genomics, and likely interest from the research community. Aim 3 - Bioinformatics component and Gene Characterization to integrate gene information from rat, mouse, and human with the data from our physiological studies. All animal models and data will be made available to the research community for further studies. PUBLIC HEALTH RELEVANCE: Hypertension and renal disease can lead to stroke, heart attack, and failure of the heart and kidneys. Genome wide association studies (GWAS) have identified potential genes and regions that are associated with high blood pressure and other complex diseases, but have provided little validation of the molecular mechanisms of these genes. The overall goal of this project is to use a novel technique to knockout genes in hypertensive and normotensive animal models to test the role of these genes in the vascular and renal mechanisms controlling blood pressure. This unique strategy will provide a mechanistic understanding of the pathophysiological role played by GWAS genes in hypertension and kidney disease.

DESCRIPTION (provided by applicant): Myocardial infarction (MI) is a major health problem in the United States. The polygenic nature of the resistance and/or sensitivity of the heart to ischemia are well accepted. Genome wide association studies and linkage analyses in both human studies and animal models have revealed a large number of chromosomal loci involved in coronary artery diseases (CAD) and MI. Unfortunately, little progress has been made in identifying causal polymorphisms directly related to the response to ischemic injury. In contrast to human studies, animal models provide the ability to identify the complex interactions through the use of specific genetic models with divergent phenotypes for myocardial ischemia such as the sensitive Dahl Salt-Sensitive and resistant Brown Norway strains. To dissect this complexity accordingly we propose: 1. Identify a gene on rat chromosome 6 responsible for resistance to ischemia. We will focus our initial positional cloning efforts on the SS.BN6 minimal congenic encompassing 3.9Mb containing 36 genes. The significance of this aim is that we are very likely to identify and validate the causal mutation in this interval. The use of engineered heart tissue (EHT) to accelerate discovery and enhancing our ability to study the mechanistic properties of the mutation is innovative. 2. Pursue the identification of the genes responsible for resistance to ischemia on rat chromosomes 3 and 12. Using SS.BN3 and SS.BN12 consomics we have already generated congenics and demonstrated that we can use our in vitro (Langendorff) and EHT models to pursue loci on these two chromosomes. Moreover, utilizing another two strains/chromosomes will lead to better understanding complexity of myocardial ischemia. 3. Functional validation of the gene(s) responsible for the resistance to ischemia. The gold standard for proving that a gene is causal requires some form of rescue experiment. We will deploy a transgenic rescue approach to validate the chromosome 6 locus. The significance of this aim is proving a mutation is casual and uses innovative solutions to generate the transgenic rescue animals. Finally, the animal models will be made available to the research community for further studies. PUBLIC HEALTH RELEVANCE: Cardiovascular disease is one of the leading causes of death worldwide and is responsible for 45% of deaths in the Western world and 24.5% of deaths in the developing countries. Myocardial infarction remains a major cause of morbidity and mortality despite anti-atherosclerotic therapies, reperfusion strategies, and anti-platelet treatment, due in part to the large heterogeneity in the response to ischemia among patients. The overall goal of this project is to identify genes and mechanisms involved in resistance to myocardial infarction.

DESCRIPTION (provided by applicant): Using the zinc finger nucleases (ZFN) technology for making gene modification or edits we generated 101 strains within two years. The ZFN technology is extraordinarily efficient and does not require the use of embryonic stem cells, as a result any rat strain should be able to be rapidly (3 to 6 months) modified. This R24 resource grant enables our existing infrastructure: personnel, expertise in gene editing, high throughput phenotyping, distribution channels resulting in an economies of scale that can be leverage to generate novel and important rat models to more investigators. The unique rat models already developed by our existing program, have demonstrated: 1) that the infrastructure is already in place to meet our objectives; 2) these gene modified strains can be used to validate a gene underlying a quantitative trait locus, testing a GWAS result, follow-up studies to define function of a predicted gene, or developing a model to study the physiology/mechanisms of a gene; 3) our economy of scale; 4) a full distribution system in place; and 5) over a decade of resources development (data, rats, genetic and genomic reagents) for the rat. To assure the resource meets community needs, we will establish an External Advisory Board (EAB) that will assess our progress, and conduct a scientific merit review process to select the genes and use of the resources of the R24 nominated by the scientific community (Aim 1). We plan to generate 250 gene targeted rat models (Aim 2), to provide a menu of phenotypic characterization that are available, but not required (Aim 3), and cryopreserved sperm from each line, along with a tissue and fibroblast primary culture bank for each strain, as well as distribute all the animals and reagents to the community (Aim 4). Specific aims 1-4 are expected to generate a cost effective and powerful collection of rat models for the community that will accelerate discovery and increase the field's understanding ofthe mechanisms involved in complex diseases. The scientific community has already nominated more than 343 genes demonstrating that the resources developed in this R24 will be of significant value to many investigators. The EAB will assure transparency and that this resource serves the community. (End of Abstract)

DESCRIPTION (provided by applicant): During the past grant cycle, using a variety of rat models, we have proven that Rab38 is responsible for the quantitative trait locus (QTL) named Rf-2, identified and validated that Sorcs1 is responsible for the QTL named Rf-1, and have identified that Shroom3 causes changes in glomerular permeability. We have made considerable strides towards cloning by position two other QTL, Rf-3 and Rf-4. In collaboration with the Chronic Kidney Disease Genetics (CKDGen) Consortium, we found that SORCS1 and SHROOM3 are significantly associated with renal disease in humans, demonstrating that this work has clinical relevance. Our long-term goal is to define the genetic architecture of end stage renal disease (ESRD). Our data suggests that a defect in both the glomerular permeability and protein trafficking in the proximal tubules is required for proteinuria, which is now our centrl hypothesis. The proposed Specific Aims are to 1) test a collection of human sequence variants of SHROOM3 predicted to be dysfunctional; 2) identify the genes/genomic elements underlying the QTL Rf-3 (interval size 1.81 Mb) and QTL Rf-4 (interval size 0.9 Mb) participating in glomerular permeability, which is thought to initiate proteinuria; and 3) test the central hypothesis that dysfunction in both glomerular permeability and protein trafficking in the proximal tubules are participating together in the development of proteinuria. Testing this central hypothesis requires the use of whole animals and cannot be done in human. Our genetic/genomic infrastructure for this proposal includes: complete genomic sequence of parental strains, NextGen sequencing tools including bioinformatic analysis, narrow congenic strains, three identified causative genes, as well as detailed physiological characterization creating an ideal platform for discovering important new genes and testing this new central hypothesis. We will study human variants of SHROOM3 in HEK293 cells, zebrafish and humanized sensitized FHH rat model for CKD. Specifically, Aim 1 tests a collection of human variants for renal disease and demonstrates how model systems can be used to follow-up genes nominated by many genome-wide association studies (GWAS). Our collaboration with the CKDGen enables us to test if the genes identified for Rf-3 and Rf-4 contribute to human disease; thereby, providing knowledge about the disease process in humans. The rationale for the proposed research is the test of human variation in SHROOM3, identification of new genes, and a new hypothesis which will provide new insights into this disease process and could identify new targets for future treatment of renal disease with a rising incidence and limited effective treatments. Specific aims 1-3 are expected to reveal the knowledge about how mutated SHROOM3 functions and identify two new genes, which will either link to known pathways or unmask new ones. We will also determine if two hits (one in the glomeruli and one in proximal tubules) are involved. Each aim of this proposal will provide fundamental advances toward defining the genetic architecture of proteinuria which is often the precursor of ESRD.

DESCRIPTION (provided by applicant): This proposal focuses on creation of a sequencing core for the Undiagnosed Disease Program (UDP) as well as comparison of the utility of genome-wide sequencing (GWS; also known as whole genome sequencing) versus Whole Exome Sequencing (WES) for the identification of causal variants. Illumina and the Medical College of Wisconsin (MCW) have worked together to advance genomic sequencing into clinical medicine; this proposal is joint between these entities. All of the necessary components for the UDP sequencing core are functional at MCW and Illumina and required capacity and turnaround are met. Both groups have championed GWS as opposed to WES for genetic discovery leading to the second focus; comparison of GWS and WES for diagnostic success. MCW uses both WES and GWS; along with obvious advantages in detecting non protein coding variants, we find significantly better coverage of actionable genes with GWS, and a higher diagnostic success rate. We thus propose to conduct GWS for all participants enrolled in the UDP creating the opportunity to compare utility of WES versus GWS. With an integrated team and using innovative lab and bioinformatics techniques we propose to test the hypothesis that GWS will produce at least 25% more diagnoses than WES. Aim 1 will generate clinical grade GWS for all UDP cases sequenced and perform read mapping and variant calling. Sharing of the data generated and the methods developed will enable the UDP network to directly compare diagnostic use of WES and GWS. Aim 2 will undertake clinical grade tertiary analysis of the data using our clinically validated analysis platform; we will also provide clinical interpretation and report generation for all cases requested. These will be produced using our existing clinical methodology and tools. Aim 2 will also support dissemination of the methodology and offer tertiary analysis and clinical interpretation to all UDN sites. Aim 3 will confirm the NextGen sequencing results using Sanger and, through gathering of this data, determine whether this step will be necessary in the future. We envision that all of the laboratory operations, methodologies, and tools developed will be made available and will be suited for cloning in additional currently non network hospitals and large clinics. Relevance: This application is highly relevant in that it seeks to establish MCW as the sequencing core for the UDP. In addition to meeting this goal, the application seeks to extend the UDN benefit by determining whether application of GWS as compared to WES provides a diagnostic advantage.