1999 — 2002 |
Krahe, Ralf |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Core--High Throughput Genotyping and Genetic Linkage Analysis @ University of Texas Md Anderson Can Ctr
The broad, long-term objectives of the High Throughput Genotyping and Genetic Linkage Analysis Facility (Core E) are to provide the members of the program and their projects with a state-of-the-art facility for high throughput, automated genotyping and genetic linkage analysis for the mutation epidemiology of childhood tumors. The specific aims of Core E are: (1) to genotype constitutive DNA samples for genome wide linkage analyses, (2) to allelotype on a targeted, genome-wide basis matched normal and tumor DNA samples for possible microsatellite instability (MSI), (4) to provide sequence analysis for TP53 mutations on DNA samples from paraffin embedded fixed tumor tissues, using the p53 GeneChip, (5) to maintain a functioning genotype database, and (6) to conduct genetic linkage analysis on the obtained genotype data. To this end, human genomic DNA samples of individuals from cancer families ascertained by Dr. Strong (Projects 1 and B) segregating either soft tissue sarcomas (STS) or osteosarcomas (OST) that have been shown to be negative for mutations in the TP53 tumor suppressor gene (Project 4), or Wilms tumor (WT, Project 2), will be analyzed using fluorescent technology and optimized panels of highly informative microsatellite markers. In addition, we will identify samples exhibiting MSI for Dr. Siciliano's (Project 5) study on genome instability and pass on him possible MSI information for quantitative analysis of this phenomenon. Similarly, mouse genomic DNA samples from inbred transgenic mice, which have been generated by Dr. Lozano (Project 4) and serve as a suitable murine model for human cancers, will be systematically genotyped. The obtained human genotype data will be analyzed for genetic linkage by complementing parametric and non- parametric linkage analysis. For the linkage analyses, Core E will interact closely with the main Linkage Analysis Facility in the Informatics and Analysis Core (Core C),, which is headed by Dr. Amos. The ultimate goals are to map the cancer susceptibility/tumor suppressor gene(s) underlying the observed increased segregation of certain cancers in the families studied by Drs. Strong (Projects 1, Core B), Huff (Project 2), and Lozano (Project 4), and to identify in the transgenic inbred mice a modifier gene for cancer susceptibility, which has been uncovered by Dr. Lozano (Project 4) and termed modifier of p53 (mop1). In addition, Core E will assist Dr. Huff (Project 2) in the identification of additional WT susceptibility gene region(s) by providing L0H information.
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0.928 |
2001 — 2005 |
Krahe, Ralf |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular Genetic Characterization of Myotonic Dystrophy @ University of Texas Md Anderson Can Ctr
DESCRIPTION (provided by applicant): The myotonic dystrophies (DM) are now collectively recognized as a clinically and genetically heterogeneous group of neuromuscular disorders, characterized by autosomal dominant inheritance, muscular dystrophy, myotonia, and multi-system involvement. Recent work by others and us indicates at least two more DM loci in addition to DM1, the (CTG)n expansion in chromosome 19q13.3. Multiple families with clinically variable presentation from predominantly distal to exclusively proximal muscle involvement show linkage to a locus in 3q21, designated DM2. However, several families with similar presentations have been excluded from this region. Thus, there is at least a third DM locus (DM3), which has yet to be mapped. The long-term goal of this proposal is the identification of DM2 in the families mapping to 3q21, and the mapping and cloning of the remaining gene(s) in the DM2-unlinked families. The characterization of the underlying mutations will be the basis for phenotype/genotype correlations. Three specific aims are proposed: (1) to clone and characterize DM2 in 3q21; (2) to clone and characterize the gene(s) in DM2-unlinked/DM3 families, and (3) to globally expression profile DM muscle with DNA microarrays. Collaborating with clinical groups from the USA and Europe, we have ascertained 57 families with clinically similar phenotypes, which are negative for the DM1 (CTG)n expansion or any of the other known myotonia loci. Ten of 21 families suitable for linkage analysis show linkage to 3q21, while 11 are unlinked. We have substantially narrowed the DM2 critical region, generated a physical transcript map and started to examine functional-positional candidate genes, using various mutation detection assays. For DM3 we propose the same strategy of positional cloning that has proved successful for DM2. Genome-wide expression profiling of DM muscle will identify dysregulated genes and provide valuable functional clues about potential candidate genes and complex cellular candidate pathways, the overall pathophysiology of DM, and potential molecular therapeutic targets. The identification of these novel genes and the characterization of their mutations and pathophysiological role(s) are the first step in developing potential therapies for patients suffering from these inherited myotonic dystrophies. Moreover, as the cellular pathologies among the different myotonic dystrophies show considerable overlap, the identification of the genes underlying DM2 and DM3 and their corresponding expression profiles may also provide valuable insights into the pathology of DM1, which continues to elude researchers.
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1 |
2006 — 2010 |
Krahe, Ralf |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
High-Throughput Genotyping and Genetic Linkage Analysis Facility @ University of Texas Md Anderson Can Ctr
The broad, long-term objectives of the High-Throughput Genotyping and Genetic Linkage Analysis Facility[unreadable] (Core E) are to provide the members of the program and their projects with a state-of-the-art facility for high-throughput,[unreadable] automated genotyping and genetic linkage analysis for the mutation epidemiology of childhood[unreadable] tumors. The objectives of Core E are: (1) to genotype constitutive DNA samples for genome-wide[unreadable] linkage and association studies, (2) to allelotype on a genome-wide or targeted basis matched[unreadable] normal/tumor DNA sample pairs for tumor-specific loss of constitutive heterozygosity (LOH), (3) to[unreadable] maintain a functioning genotype database, and (4) to conduct genetic analyses on the obtained[unreadable] genotype data. To this end, human genomic DNA samples of individuals from cancer families ascertained[unreadable] by Dr. Strong (Core B) segregating either soft tissue sarcomas (STS) or osteosarcomas (OST) and[unreadable] classified as having Li-Fraumeni syndrome (LFS) or one of its variants (Dr. Strong/Project 1 and Dr.[unreadable] Krahe/Project 2), or Wilms' tumor (Dr. Huff/Project 4) will be analyzed using various complementing high-throughput[unreadable] technologies integrating microsatellite and single nucleotide polymorphism (SNP) markers to[unreadable] identify genomic regions co-segregating with the disease (Projects 1 and 2) and to confirm regions of LOH[unreadable] (Projects 2 and 4). Genotyping platforms include fluorescent technology with highly informative[unreadable] microsatellite markers in optimized panels for genome-wide genotyping and custom markers using a[unreadable] universal primer approach for regional fine mapping (Projects 1, 2, and 4). High-density SNP microarrays[unreadable] (approximately 10,000 or about 100,000 SNPs) will be used to genotype individuals in both p53 and non-p53 families to identify modifier genes of tumor susceptibility (Projects 1 and 2) and to allelotype matched normal/tumor DNA[unreadable] sample pairs to identify regions of tumor-specific loss of constitutive heterozygosity (Projects 1 and 2).[unreadable] Pyrosequencing will be used to type additional SNP markers in targeted regions identified by the above[unreadable] approaches for fine mapping (Projects 1, 2, and 4). The obtained human genotype data will be analyzed for[unreadable] genetic linkage and association by complementing parametric and non-parametric analysis methods. For[unreadable] these analyses, Core E will interact closely with the Statistical Genetics and Bioinformatics Core (Core C)[unreadable] headed by Dr. Amos. The ultimate goals are to map major cancer susceptibility genes and modifier genes[unreadable] that underlie the observed increased segregation of certain cancers in the families studied by Drs. Strong[unreadable] (Projects 1, Core B), Krahe (Project 2), and Huff (Project 4), to map and identify modifiers of tumor[unreadable] susceptibility (Projects 1 and 2), and to identify additional genomic regions that may be involved in tumor[unreadable] development and/or progression by providing LOH information (Project 2).
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0.928 |
2006 — 2010 |
Krahe, Ralf |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Molecular Genetic Anatomy of Li Fraumeni Syndrome (Lfs) @ University of Texas Md Anderson Can Ctr
Li-Fraumeni syndrome (LFS) is a clinically and genetically heterogeneous inherited cancer syndrome. Most[unreadable] cases (approximately 70%) identified and characterized to date are associated with dominant germline mutations in the[unreadable] tumor suppressor gene TP53 (p53). Another tumor suppressor gene, CHEK2, was recently identified as a[unreadable] second minor predisposing locus. Studying a series of non-p53 LFS kindreds, we have shown that there is[unreadable] additional genetic heterogeneity in LFS kindreds with inherited predisposition at a locus other than p53 or[unreadable] CHEK2. Using a genome-wide scan for linkage with complementing parametric and non-parametric analysis[unreadable] methods, we have identified linkage to a separate, previously not implicated genomic region. In addition to a[unreadable] major predisposing locus, there is evidence for significant heterogeneity in risk within and between kindreds,[unreadable] in both p53 and non-p53 LFS kindreds. These data implicate additional risk modifiers in the genesis of LFS[unreadable] and its variants, including another major gene(s) as well as modifier genes. We hypothesize that the[unreadable] inherited susceptibility to childhood and associated cancers in non-p53, similar to p53, LFS kindreds[unreadable] is the result of a highly penetrant, dominantly acting gene. In keeping with a multi-step carcinogenesis[unreadable] model, however, germline mutations are not sufficient, and other modifier genes and factors,[unreadable] including epigenetic alterations, appear to be necessary in both p53 and non-p53 LFS kindreds. To[unreadable] test these hypotheses, we propose the following three specific aims that take advantage of the unique and[unreadable] large resources assembled as part of this program project: (1) to identify and characterize the gene for the[unreadable] newly mapped non-p53 LFS susceptibility locus; (2) to identify p53 and non-p53 LFS modifier genes;[unreadable] and (3) to evaluate the contribution of promoter hypermethylation and transcriptional inactivation of[unreadable] known cancer genes subject to epigenetic silencing to the LFS phenotype. Identification of the major[unreadable] non-p53 predisposing gene and its underlying mutations should provide insight into other genetic events that[unreadable] predispose to the genesis of diverse tumor types associated with LFS and its variants. Our integrated[unreadable] genomics approach that combines genomic and transcriptomic with epigenomic profiling will yield a better[unreadable] understanding of the complex molecular genetic and epigenetic events underlying the multi-step[unreadable] carcinogenesis in LFS and its variants and will provide valuable functional clues about potential candidate[unreadable] cancer and modifier genes, complex cellular candidate pathways, and the overall pathophysiology. As a[unreadable] childhood cancer, LFS is a unique model to study the underlying genetic events associated with a complex[unreadable] cancer syndrome, presumably because fewer such alterations are needed to give rise to the associated[unreadable] cancer. Similar to p53, other LFS predisposition and/or modifier genes may be functionally similarly important[unreadable] in other solid tumor types lacking a clear predisposition and inheritance pattern.
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0.928 |
2012 — 2013 |
Krahe, Ralf |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Role of Znf9 in Pathogenesis of Myotonic Dystrophy Type 2 (Dm2) @ University of Tx Md Anderson Can Ctr
DESCRIPTION (provided by applicant): Myotonic dystrophy (DM) is the most common adult-onset muscular dystrophy. Presently there is no effective treatment and, although the mutations are known, the pathomechanisms are incompletely understood. DM1 and DM2 are caused by different expansion mutations--(CTG) DM1 in DMPK and (CCTG) DM2 in ZNF9. Despite similar mutations, they are clinically distinct diseases. Since transcription of (CUG) DM1/(CCUG)DM2 is necessary and sufficient to cause disease, DM1 and DM2 are considered toxic RNA diseases. However, the only mechanism of toxicity extensively studied to date is aberrant splicing, and therapeutic efforts have focused mainly on this aspect. What is not known is the extent to which missplicing accounts for the symptoms and whether there are additional pathomechanisms. This knowledge gap limits understanding of the effectiveness of therapies that target missplicing. Our long-term goal is to understand the pathomechanisms underlying DM and to identify entry points for therapy. Evidence from animal models, particularly Znf9 mice, suggests that many DM2 features can be elicited by ZNF9 insufficiency, without (CCTG)/(CCUG)DM2 or aberrant splicing. We and others have demonstrated reduced ZNF9 mRNA and protein levels in DM2 patients. Therefore, ZNF9 loss is directly implicated in DM2 pathogenesis, but its role remains unknown. Our central hypothesis is that expression of (CCUG)DM2 in ZNF9 is toxic via multiple mechanisms. The objective of this application is to determine the role of ZNF9 in the pathology of DM2. The rationale for the proposed research is that since ZNF9 functions as a regulator of both transcription and translation, ZNF9 haploinsufficiency plays a critical role in DM2 by dysregulation of both transcriptional and translational targets. Because in mice ZNF9 haploinsufficiency alone results in a DM-like phenotype, we hypothesize that mutations in human ZNF9 can produce DM2-like symptoms. To test our hypotheses, we will pursue three specific aims: (1) Identify ZNF9 DNA binding sites and transcriptional targets; (2) Identify ZNF9 mRNA binding sites and translational targets; and (3) Sequence ZNF9 in patients with DM of unknown etiology, to identify mutations responsible for their DM-like phenotype. Aims 1 and 2 will utilize Next-Generation technology to sequence ZNF9- immunoprecipitated DNA and RNA complexes to identify ZNF9 targets. Candidate genes will be validated in muscle biopsies and cultures using RNA- and protein-based methods. In Aim 3 we will sequence ZNF9 in an already collected DM patient cohort without (CTG) DM1/(CCTG)DM2 expansions. The approach is innovative, because it utilizes global technologies and unique patient resources to investigate previously unrecognized pathomechanisms. The proposed research is significant because identification of a pathomechanism(s) independent of aberrant splicing will shift the existing paradigm, focused on splicing. Ultimately, such knowledge will provide new insights into the role of ZNF9 in DM2 and identify new candidate effector genes and cellular pathways as well as therapeutic targets, which will positively impact the quality of patient lives. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because it is ultimately expected to increase understanding of the role of ZNF9 and heretofore unrecognized mechanisms in the pathogenesis of myotonic dystrophy type 2 (DM2). Successful completion will result in the identification of new candidate effector genes and cellular pathways as well as targets for therapeutic intervention specific to myotonic dystrophy. Thus, the proposed research is relevant to the part of NIAMS/NINDS missions that pertains to developing fundamental knowledge that will help reduce the burdens of neuromuscular disease in human patients.
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0.928 |