Joseph H. Nadeau - US grants

DESCRIPTION (provided by applicant): Many models of common human diseases and numerous traits of great biological interest vary among inbred strains of laboratory mice. Dissecting their multigenic control and identifying the responsible genes has been notoriously difficult. Chromosome substitution strains (CSSs), single chromosome substitutions on a defined and inbred genetic background, represent a novel and powerful paradigm. Proof-of-concept studies, with funds from other sources, demonstrated that these strains are remarkably powerful for detecting genetic variants that eluded detection with any other mapping method. During the previous funding period, we completed construction of the 22 CSSs in the B6.A-Chr panel. These strains were provided to the Jackson Laboratory for preservation and distribution as a resource for the biomedical research community. In addition, one A.B6-Chr CSS, and eight 129.B6 and B6.129 CSSs were completed and the remainder of these panels is under construction. We propose two Specific Aims: Specific Aim 1: Complete construction of the 129.B6 and B6.129 CSSs. Eight strains are complete and the remainder is at various stages of backcrossing. These CSSs can be used to dissect the genetic control of traits that differ between these strains, they can be used to characterize modifier genes that modulate phenotypes of mice with engineered mutations that are made with ES (embryonic stem) cells that are derived from 129/Sv mice, and they can be used to identify proteins that are critical to establish ES cell lines. Specific Aim 2: Complete construction of 10 129.MOLF CSSs. These strains will be valuable for studying traits that differ between these strains that are derived from mice that diverged more than 100,000 years ago. These strains can also be used to characterize modifier genes that modulate phenotypes in these mice. These CSS panels will revolutionize studies of complex traits by facilitating detection and discovery of QTLs.

DESCRIPTION: (Adapted from investigator's abstract): This is a revised application for a new R01 from an established investigator. The PI has requested 3 years of support to investigate the genetic basis of hyperhomocysteinemia and its relationship to vascular pathology in several inbred strains of mice. Levels of Hcy in the C57BL/6J strain are twofold higher than the A/J and C3H/HeJ strains; (C57BL/6J x A/J) F1 animals are similar (but not identical) to the A/J parent; by contrast, (C57BL/6J x C3H/HeJ)F1 animals exhibit so-called heterosis in that their levels are apparently higher than either parent. In Specific Aim I, the underlying genetic basis of these differences will be investigated by determining Hcy levels in recombinant inbred strains for each of the two pairs, C57BL/6J x C3H/HeJ, and by analyzing 320 progeny from an F2 intercross for QTLs. Hcy levels will also be examined in 21 consomic strains (congenic for each of 21 A/J chromes) in the C57BL/6J background. In Specific Aim IV, the potential relationship of increased Hcy levels to vascular pathology will be investigated by measuring lipid-containing intimal lesions in large and medium sized arteries from the RI strains and from PL/J (see below). In humans, decreased activity of methylene tetrahydrofolate reductase (MTHFR) contributes to elevated Hcy levels because its product is a cofactor for conversion of Hcy to Methionine. The PI has discovered that the PL/J strain exhibits approximately twofold lower activity of MTHFR than most inbred strains, and, in Specific Aim II, proposes to investigate the genetic basis for this difference bet determining MTHFR activity in 200 progeny from an (C57BL/6J x PL/J)F2 intercross. Finally, in Specific Aim III, the effect of age, sex, pregnancy, and folate supplementation on Hcy levels will be examined in C57BL/6J and A/J MICE, and in C57BL/6J mice that carry one of two mutations, Gli3 or ApoB, that apparently cause a secondary increase in Hcy levels by an unknown mechanism. A part of these experiments, the PI will also complete a survey of Hcy levels in MTHFR activity in additional inbred strains.

[unreadable] DESCRIPTION (provided by applicant): Testicular germ cells tumors (TGCTs) are the most common cancer affecting young men and their incidence has been rising dramatically world-wide. Genetic markers for inherited risk are needed to identify susceptible individuals for regular surveillance and early treatment. Many TGCTs arise from primordial germ cells (PGCs) during fetal development. Little is known about the genetics or biology of this pluripotent stem cell lineage. The 129 family of inbred strains are the only strains in which spontaneous TGCTs occur at an appreciable frequency (5%). Several single gene mutations such as Ter, Ay and SI modulate susceptibility on the sensitized 129 genetic background. These strains and mutants are the foundation for genetic and developmental studies of PGC development and TGCT susceptibility. During the previous grant period, we showed that (a) Ter, the most potent TGCT modifier known, results from a mutation in the Deadend gene, (b) the TGCT suppressor at the Ay locus is Raly or Eif2b2 but not agouti, and also that the MGF enhancer is located in a 120 kb interval in which MGF is the only conventional gene, and (c) several single modifiers interact to modulate TGCT susceptibility, including dramatically reduced susceptibility in p53/+ SIJ/+ double mutant mice. [unreadable] [unreadable] We propose four Specific Aims: [unreadable] [unreadable] Specific Aim 1. What is the identity of the TGCT suppressor in Ay mutants and the enhancer in Kitl*SI mice? [unreadable] [unreadable] Specific Aim 2. What is the nature of the interactions between TGCT susceptibility modifier genes? [unreadable] [unreadable] Specific Aim 3. Does the Y chromosome affect TGCT susceptibility? [unreadable] [unreadable] Specific Aim 4. Do mutations in RNA editing genes affect TGCT susceptibility? [unreadable] [unreadable] Our discovery that the most potent TGCT modifier gene (Ter) probably involves anomalies in RNA editing raises exciting new opportunities to explore the role of this remarkable but poorly understood process in stem cell biology and TGCT susceptibility. [unreadable] [unreadable] [unreadable]

DESCRIPTION: (Adapted from investigator's abstract) Disorganization (Ds), a single gene mouse mutation, has profound effects on mouse development. Unlike most mutations where particular traits with diagnostic features are inherited among generations, the heritable trait in mice with the Ds mutation is the propensity for the body to be formed in an unpredictable manner. No two mice are affected in identical ways. Affected mice may show agenesis, duplication or fusion of structures, and all morphological structures seem prone to anomaly. Remarkably, these mice are not prone to cancer, spotting, behavior, and longevity or fertility problems. Ds is one of the few examples in mammals of a true dominant, gain-of-function mutation. Determining the identity of the Ds gene is important because deep insights into the regulation of pattern formation during development can be gained. In addition, insights into the molecular nature of mutations that show variable expression and low penetrance may be forthcoming. Ds has been located to a 0.2 cM segment of mouse Chr 14, cloned in a 360 kb BAC contig and 80 kb of sequence has been obtained. Cloning of the mouse Ds gene is proposed. The genomic sequence of the Ds locus will be determined in wild-type mice in order to identify genes and markers, evidence for rearrangement in Ds will be sought, cDNA expression and sequence will be assessed in wild type and Ds mice, and the genomic sequence of the Ds locus will be determined. In addition, it is proposed that the identity of Ds will be verified by generation and characterization of transgenic mice with the candidate Ds mutation.

Attributing function to genes, their regulatory elements and other functional DNA sequences will be the next step in genomics. This functional phase involves researchers from a variety of disciplines, including computer science and engineering. To facilitate the use of genomic informatin in function studies, pathways will be used to present the information. Pathways require detailed integration of diverse sources of information. Researchers will need transparent access that will be provided through this project. The project will integrate the data from a pathways rather than a sequence perspective, enabling scientists who do not do the sequencing to add functional information. New tools will enable users to visualize pathways data at mutiple levels of detail and abstraction. The use of XML will permit dissemination of query outputs for control of the data. Metabolic pathways, transcriptional regulation and protein synthesis pathways and signal transduction pathways will be the first incorporated in this web-based system. The research provides both for student support in both biology and computer science and a resource for the functional genomics community.

Case Western Reserve University has been awarded a grant to hold a workshop from September 28th-October 3rd, 2003 on the Island of Santorini in Greece with the aim of bringing together a very broad group of researchers to integrate their very disparate data sets to solve some very basic and fundamental problems in biology. Specifically, the main focus will be resolving a model for looking at the multidimensional nature of protein function in time, space and context. A large part of this involves specifically determining how to combine disparate sets of data on sequence, structure, pathways, networks and function. The conference will include many young and early career researchers.

Context Statement
(Sensor and Sensor Networks Panel, June 12 and 13, 2003)

Approximately 930 proposals were submitted in response to the Sensors and Sensor Networks Solicitation (NSF 03-512) during fiscal year 2003. A total of 469 of these proposals were considered in the Small-Team competition. All proposals were grouped according to their relevance to one of the three general topical areas identified in the solicitation. This proposal is one of 30 considered in a panel on June 12-13, 2003 on the subject of Designs, Materials and Concepts for New Biological Sensors and Sensing Systems. This panel was jointly run by program directors from the Experimental and Integrated Activities Division of CISE and the Bioengineering and Environmental Systems Division of ENG. The panel included sixteen panelists, all technical experts, who were invited to NSF and who reflect the range of expertise needed for the proposals under consideration. The panelists had reviewed the proposals in their areas of expertise, and sent their individual reviews via FastLane, prior to coming to the panel meeting. Proposals were reviewed and evaluated against both merit review criteria established by the National Science Board, namely, "What is the intellectual merit of the proposed activity?" and "What are the broader impacts of the proposed activity?" In addition, the proposals were assessed for relevance to the Program Solicitation (NSF 03-512).

At least three panelists provided written evaluations for each proposal. The written evaluations were presented to the panelists who debated the strengths and weaknesses of the proposals, which were then assigned preliminary ratings. The panel discussions concerning the proposals were documented by a panel Recorder, who submitted the summary of the discussion to the panel for unanimous approval. After all the proposals had been reviewed and rated, the panel placed the proposals into two categories: (1) Recommended and (2) Not Recommended for funding. The panel provided sufficient information for the Program Director to make a recommendation.

[unreadable] DESCRIPTION (provided by applicant): Birth defects occur frequently in humans but occurrence in most cases is sporadic without obvious genetic or environmental etiology. The mouse mutation Disorganization (Ds) is an extraordinary example of a single gene Mendelian trait that has sporadic patterns of occurrence because of its exceptional genetic and developmental features. Ds is one of the rare examples of a trait that is inherited in a true-dominant and gain-of-function manner. Its developmental attributes are even more striking. Ds causes an extraordinary variety of birth defects involving most body parts, but remarkably no two mice are affected in the same way. The defects are always asymmetrical but often show mirror-image symmetry in the affected structure. The nature of the various birth defects suggests both that the Disorganization gene plays a fundamental role in pattern formation and lineage determination during embryonic development, and the nature of the mutation leads to unpredictable anomalies in the normal patterns of development. During the previous funding period, we discovered a new insertion of an ETnII transposable element at the Ds locus, and a BAC with this element and three flanking genes that can cause Disorganization-like birth defects in transgenic mice. However, the location of this ETnll insertion between genes suggests that it did not disrupt coding sequences, but instead may cause chimeric transcripts, it may adversely affect the expression of flanking genes, or the insertion may have disrupted a non-coding functional element. To identify the Ds gene, we propose Specific Aim 1: to complete the comparison between the finished sequence in Ds mice with the corresponding sequence in the DA/Hu strain on which the Ds mutation arose; Specific Aim 2: to test expression patterns (chimeric transcripts and mRNA levels) of the three candidate genes during embryonic development in DS/Ei and DA/Hu embryos; and Specific Aim 3: to test for affected mice in BAC transgenics, ETnll knock-in mutants at the Ds locus in wild-type mice, and the functional consequences of ubiquitous over-expression of each of the three flanking genes in wild-type mice. These systematic studies should lead to identification of the Disorganization gene and to insights into the control of fundamental developmental processes and the ways in which sporadic birth defects arise. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): We propose small, intensive annual workshops whose mission is to coordinate mouse genetic, genomic, biological and bioinformatics research and resources towards the 10-year goal of functionally annotating the DNA sequence of the mouse genome. This goal will be accomplished by (1) producing at least one heritable mutation in ES cells, mice or both in every gene in the genome; (2) identifying every gene that affects key traits of biomedical interest; (3) establishing an infrastructure for preserving and distributing mutant cells, mice and gametes; and (4) enhancing the informatics and database support for these functional studies. The workshops will involve invited academic and commercial experts from the United States, Canada, Europe, Japan and Australia. The specific aims of the workshops are - To coordinate the production, characterization, archival and distribution of ES cells, gametes and mice; - To develop 2nd generation phenotypic assays for novel functional variants by working with experts in particular biological areas; - To assess progress, problems and solutions that are being addressed in the large and small mutagenesis programs; - To coordinate the management of genetic and phenotypic information about these mutant mice in a centralized, publicly accessible database; - To evaluate the usage and utility of mutant mice by the larger biomedical research community.

This award provides support for students and young researchers to attend the 4th Pathways, Networks and Systems International Conference to be held October 8-13, 2006 at the Royal Myconian Conference Center, Mykonos, Greece. The conference uniquely blends experimental studies of complex multilevel systems with rigorous computational, bioinformatics, statistical and analytical approaches. Emphasis is on mammalian systems, but breakthrough discoveries in other species are included where they lead to new strategies, paradigms, analytical methods, and phenotyping methods. Attendance is limited to less than 100 to promote interactions. The conference is a balanced mix of formal and long platform presentations with ample time for informal discussions. Women, students, fellows and underrepresented individuals are encouraged to attend, will receive financial support from this award and placement in the presentation program. Feedback from previous conferences has been exceptional. Although there are other genomics, computational and systems biology conferences, none integrate computational and experimental so seamlessly or effectively.

This award provides partial support for travels of students, young faculty, and especially members of underrepresented groups from the US institutions, to attend the 5th Pathways, Networks and Systems International Conference in Porto Heli, Greece, 24-29 June 2007. The conference uniquely blends experimental studies of complex multilevel systems with rigorous computational, bioinformatics, statistical and analytical approaches. Emphasis is on mammalian systems, but breakthrough discoveries in other species are included where they lead to new strategies, paradigms, analytical methods, and phenotyping methods. Attendance is limited to less than 100 to promote direct and personal interactions. The conference is a balanced mix of formal and long (45 min) platform presentations with ample time for informal discussions. Feedback from previous conferences has been exceptional. Although there are other genomics, computational and systems biology conferences, none integrate computational and experimental approaches so seamlessly or effectively.

DESCRIPTION (provided by applicant): Support is requested for the 7th Pathways, Networks and Systems Medicine Conference. The importance of understanding health and disease from a systems perspective is increasingly recognized as a high priority complement to the traditional reductionist paradigm. In particular, a deep understanding of the functions of every component in complex biological systems must be fully integrated with multilevel systems analysis. The functions of these components make sense only in the context of systems properties, and similarly systems are meaningful only with a firm molecular foundation. In each case, the consequences of genetic variation (mutations and polymorphisms) as well as environmental factors (e.g. diet, drugs and microbes), which act as systems perturbations, must be included in order to reflect the natural circumstances of the human condition. This complementary integration of components and systems is one of the primary challenges in biomedical research, and it is the focus of these Conferences. The Pathways, Networks and Systems Medicine Conferences uniquely blend experimental studies of complex multilevel biological systems with rigorous computational, bioinformatics, statistical and analytical approaches. Emphasis is on mammalian systems in order to maximize the relevance to human disease. However, breakthrough discoveries in other species are included where they lead to important systems principles, research strategies, analytical paradigms, and phenotyping methods. Session topics focus on (1) cancer, (2) metabolism and cardiovascular systems, (3) stem cells, development and regeneration, and (4) immunity and inflammation. Attendance is limited to less than 100 to promote interactions. The conference is a balanced mix of formal, platform presentations with ample time for informal discussions. Women, students, fellows, individuals from groups who are traditionally under-represented in science, and persons with disabilities are encouraged to attend, and receive preferential financial support and placement in the presentation program. Greece provides a central location for an international conference and Corfu an isolated location that encourages individuals to remain for the entire conference. Feedback from previous conferences has been exceptional, - 'best conference ever in every way', 'I don't want the conference to end, the science is so good', and 'the most important conference I've attended in 30 thirty years'for example. Although other conferences focus on related genomics, computational and systems issues, none focus explicitly on systems medicine in humans and mammalian models, and none integrate computational and experimental studies as seamlessly and effectively. (End of Abstract)

Failure of neural tube closure is a complex genetic disorder, rarely if ever seen as a simple Mendelian trait. In the clinical population, NTDs are probably most often caused by partial inactivation and/or gain of function mutations of multiple genes, acting in conjunction with the gestational environment. Recognizing such changes as disease associated mutations rather than simple polymorphisms is a challenge for assessment of risk in individual families. Another challenge is determining the most appropriate means to reduce NTD occurrence for a particular couple. Recent studies from these three collaborating laboratories lead us to hypothesize that a systematic examination of gene network interactions in mouse models will elucidate patterns that are applicable to risk assessment and prevention of NTDs in humans. In a mouse line with a folic acid (FA) responsive NTD, we identified a point mutation in Lrp6, a co-receptor for Wnt canonical signaling, which implicates several important developmental genetic pathways likely to be encountered clinically. Using this Crooked tail (Cd) mouse, we identified a possible gene expression and metabolism "signature" in liver tissue and blood, predicting a genetic make-up that will respond to FA supplementation by preventing NTD. In addition, we used ENU mutagenesis screens to identify NTD associated mouse mutations that provide candidates for human testing and which can be tested for response to FA and other potential NTD preventive agents. We propose to pursue a multi-institution effort to characterize genetic susceptibility profiles in mice and identify genetic backgrounds that respond to prevention measures such as dietary supplementation. Experiments will use FA and inositol supplementation to test whether these agents prevent NTD in Lrp6 null and NTD-prone mice with defined ENU-generated mutations. Gene transcript arrays will examine expression in the neural tube during closure in these mutants using cluster analysis to compare patterns in the mutant vs. wildtype siblings. In addition, the genetic and metabolic signature identified in Cd non-neural tissues will be compared against signatures in other ENU-generated and naturally occurring NTD mouse mutants to determine the predictive power of this pattern. This project will build a framework for rational approaches to new treatments based on molecular pathways. To this end, the relationship between the biochemical pathway that is disrupted and the responsiveness or resistance to FA supplementation will be examined in several mouse lines. This will begin to reveal mechanisms by which FA, and inositol, acts to suppress NTD.

Traditionally genetics has focused on direct associations between genotypes and phenotypes within individuals. This logical focus on principles of Mendelian genetics has led to a revolution in our understanding of fundamental biological processes and disease genetics. However, observations such as 'missing heritability' in genome-wide association studies suggest that our explanations for phenotypic variation and disease risk are incomplete in important ways. In addition, several recent reports of interacting genes in different generations and transgenerational genetic effects strongly suggest that alternative modes of inheritance exist. These reports involve a wide variety of embryonic and adult traits and can lead to dysfunctions and diseases such as embryonic lethality, cancer, obesity and anxiety. Attributing phenotypes to gene action in previous generations is a fundamental and profound observation that suggests that both epigenetic (non-DNA) and genetic (DNA) mechanisms guide inheritance. Our discoveries implicate RNA editing, miRNA biology, translation control as well as perhaps RNA granules in these transgenerational effects. Proposed work will identify the molecular basis for transgenerational effects and characterize the mechanisms for epigenetics across generations. My accomplishments in comparative genomics, the developmental biology of neural tube defects and testicular cancer, the physiological genetics of obesity and metabolic diseases, and the genetic architecture of complex traits demonstrate my ability to identify hard problems and make important contributions in an unusually wide variety of biomedical fields. The proposed work on transgenerational genetic effects represents a new and exciting area of research, partly because it involves questions and technologies that I am eager to learn, but more importantly because the results could revolutionize our understanding of the molecular mechanisms of inheritance as well as assessment of phenotypic variation and disease risk.

DESCRIPTION (provided by applicant): Testicular germ cell tumors (TGCTs) are the most common cancer affecting young men, and the incidence has increased dramatically in the last 50 years. The genetic control of susceptibility is unusually complex, and with a single exception genetic variants that control susceptibility elude discovery in humans. We discovered the identity of three genetic modifiers that modulate susceptibility in a mouse model. Deadend1*Ter, which increases susceptibility, is related to genes involved in RNA editing and blocks access of specific miRNAs to their target mRNAs. Similarly, we showed that partial deficiency for the Eif2s2 translation initiation factor at the agouti-yellow locus suppresses susceptibility. Finally, we showed that loss of the transmembrane but not the soluble isoform of the Kit ligand increases susceptibility in mice. Two recent papers showed that Kit ligand is a major TCGT susceptibility gene in humans. Together these results suggest that interactions between 5'cap and 3'UTR in target mRNAs modulate global and perhaps transcript-specific translation. TGCT stem cells appear to be unusually sensitive to changes in translation rates. With these important discoveries that TGCT modifier genes involve related functions, we can now address specific questions about mechanisms of tumorigenesis for a developmentally important stem cell lineage - primordial germ cells. In addition, we found spontaneous metastasis in several TGCT susceptible strains. TGCT metastasis occurs frequently in humans and the mechanisms are poorly understood. Our exciting discovery therefore enables unique studies of the mechanisms of TGCT metastasis that were not previously possible. We propose Specific Aims to address four questions: (1) What are the mechanisms for TGCT suppressor effects of Eif2s2 haplosufficiency? (2) Do Dnd1 mutants affect RNA editing? (3) Do RNA editing mutants affect TGCT susceptibility? (4) What are the characteristics of putative TGCT metastases in mouse models? PUBLIC HEALTH RELEVANCE: Testicular cancer is one of the most common malignancies affecting young men. Although the genetic control of susceptibility is unusually strong, little progress has been made finding these genes that could be used define the mechanisms of susceptibility and that could serve as diagnostic markers or as treatment targets. We discovered three TGCT susceptibility genes, two of which suggest that control of mRNA translation has dramatic effects on susceptibility. In this application, we propose studies to test hypotheses about mechanisms by which changes in translation control modulate TGCT susceptibility. We also discovered, and propose to characterize spontaneous TGCT metastases in several of our mouse models.