Vivian G. Cheung - US grants

Hearing loss affects over 40 million people in the United States. It is the most common form of sensory defect in humans. The mechanism of hearing is still largely unknown despite recent advances in molecular medicine. The goal of this proposal is to apply two molecular biology techniques, genomic mismatch scanning and DNA microarray, to gain a better understanding of the genetic basis of hearing loss. This will eventually affect the prevention, treatment and care of hearing disorders. Genomic mismatch scanning (GMS) is a high-throughput genetic linkage technique that allows physical isolation of the identical-by-descent DNA fragments shared between two related individuals. In this proposal, we plan to apply GMS to confirm the localization for DFNBI, the gene for non-syndromic autosomal recessive deafness that has been mapped by linkage to chromosome 13q. Then, we will narrow the DFNBI candidate region using linkage disequilibrium mapping. This has been difficult with traditional mapping strategies due to the need for a very dense set of polymorphic markers. However, in GMS, a dense set of completely informative markers is scanned simultaneously on a whole genome level. In addition, during the grant period, we will develop a DNA microarray that will allow mapping of the identical-by-descent DNA fragments isolated by GMS on a whole genome level in one hybridization step. This DNA microarray will allow mapping of other deafness loci with unknown genomic locations. Finally, we will develop a linkage analysis model for the identity-by-descent maps generated by GMS. Upon achieving the goals delineated in this proposal, we hope to have contributed to the understanding of the genetic control of hearing and have provided a robust method that is a promising tool for the next generation of gene mapping.

Unraveling the genetic control of diseases has important impact in disease prevention, diagnosis and therapy. Positional cloning is the main strategy for disease gene mapping. Despite advances in technology and increasing resources such as semi-automated genotyping instruments and dense maps of genetic markers, gene mapping remains a difficult task. Recently, we have developed an alternative gene mapping method that does not require genotyping. Instead gene regions are isolated by genome screens for shared genomic segments between individuals affected with the disease of interest. The isolated DNA fragments are then mapped by hybridization onto a genomic microarray. We call this method direct identical by descent (IBD) mapping. It is a combination of two techniques: genomic mismatch scanning and DNA microarrays. We have shown that direct IBD mapping is feasible on one chromosome by mapping the gene for congenital hyperinsulinism. The goal of this proposal is to expand this result by accomplishing the following aims. 1. To generate a whole genome DNA microarray for gene mapping, a "genome on a chip". 2. To test genome wide IBD mapping in CEPH grandparent-grandchild pairs. 3. To develop statistical methods for the analysis of data derived from direct IBD mapping. Development of direct IBD mapping should provide a gene mapping method that is less labor-intensive and more efficient than the current methods. We expect that it can be applied as a tool for genome wide linkage disequilibrium analysis and affected relative pair mapping.

Bacterial artificial chromosome (BAC) clones are used in a wide range of projects from genome sequencing to gene discovery. Our laboratory is identifying and assembling a set of mapped human BAC clones. This resource is available for investigators who wish to use them. The BAC clones in our resource are anchored to sequence tagged site (STS) markers in radiation hybrid maps. The mapping information of the clones is freely available on our web-based database, GenMapDB. As we mapped clones to add to this resource, it became clear that the value of this collection would be greatly increased if we could further characterize the clones and ensure that the genomic location assigned to each clone is correct. In this application, we propose to achieve these goals by accomplishing the following aims. 1. To prepare DNA on all the mapped clones for further analysis including fluorescent in situ hybridization (FISH) carried out by our collaborators and our own further characterization (Aim 2). 2. To further characterize the clones by performing HindIII fingerprinting and end sequencing. 3. To upgrade the current database and to assess the accuracy of our RH-based BAC map with data from FISH analysis. The additional information about the clones will provide a genomic reagent that can be used in projects such as mapping of chromosomal mutations, gene mapping and polymorphism screening. In the process of characterizing the clones, we will also get information that gives us insight into the concordance rate between RH map, genome sequence map and cytogenetic map.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] Autosomal recessive diseases are by definition those where only individuals with two mutated copies of the disease genes are affected. However, even in these diseases, there is often some manifestation in the heterozygous carriers. While there are usually no marked phenotypes in carriers, they often have subtle phenotypes that are minor differences from non-carriers. Most autosomal recessive diseases are rare but carriers are not. All individuals are carriers of several deleterious mutations. These mutations are likely to contribute significantly to the wide variation in phenotype among us, from disease susceptibility to variation in response to stress. [unreadable] [unreadable] In this project, we will study the carriers of radiosensitivity syndromes in order to understand the individual variation in response to radiation. We will focus on the gene expression profiles of heterozygous carriers of four radiosensitivity syndromes: Ataxia Telangiectasia, Nijmegen Breakage Syndrome, Bloom Syndrome and Fanconi Anemia. Physical examination and standard biochemical tests do not reliably detect the subtle phenotypes in these carriers. Our previous work (Watts et al, 2002) establishes that heterozygous carriers of Ataxia Telangiectasia have a "gene expression phenotype." In this project, we will extend and determine whether carriers of other radiosensitivity syndromes also have expression phenotypes at baseline and in response to ionizing radiation. The specific aims are: 1) Identify the expression phenotype of carriers of Ataxia Telangiectasia, Bloom Syndrome, Nijmegen Breakage Syndrome and Fanconi Anemia at baseline; 2) Characterize the expression phenotype of carriers of Ataxia Telangiectasia, Bloom Syndrome, Nijmegen Breakage Syndrome and Fanconi Anemia in response to ionizing radiation (IR). [unreadable] [unreadable] The results from this study will have important implications for understanding the basis of variation in radiation response. The approach can also be broadened to study the contribution of heterozygosity of recessive diseases to the complex genetic architecture of human diseases and traits. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The focus of this renewal application is DNA sequence variation. Our goal is to continue to develop a mapping method known as direct IBD mapping and to study the pattern of meiotic recombination in humans. Altered recombination is associated with non-disjunction, the major cause of aneuploidy. First, direct IBD mapping is a method that allows identification of genomic regions shared between related individuals without marker-by-marker genotyping. Large DNA segments shared identical in sequence (likely representing IBD DNA fragments) between related individuals are enriched in a procedure known as genomic mismatch scanning. The selected DNA fragments are then mapped by hybridization onto a genomic DNA microarray. In the last several years, we have optimized the steps and created the necessary resources for direct IBD mapping. Next, we will validate the procedure by mapping the IBD regions shared between individuals in CEPH families and will streamline various steps so that direct IBD mapping can become a tool for high-throughput mapping. Second, stimulated by the work on direct IBD mapping, we have begun to characterize the pattern of meiotic recombination in humans. In this renewal application, we will expand the scope of the project to characterize the variation in human recombination rates and map the genetic determinants of this variation. This proposal has the following aims: 1. Develop direct IBD mapping, a high-resolution identity-by-descent mapping method that does not require genotyping. 2. Characterize natural variation in recombination rate in humans. 3. Map the genetic determinants of natural variation in total recombination rate in humans. We expect the results from this study will provide a robust mapping method that is more efficient than current methods. It will also give insights into the pattern of human meiotic recombination, a key process that contributes to genetic diversity and to risk of non-disjunction.

DESCRIPTION (provided by applicant): People are exposed to radiation through various sources. The use of radiation for diagnostic and therapeutic purposes is increasing. While these procedures bring significant benefits, radiation exposure carries risks. Prediction of risk from radiation exposure can be improved by taking into account the different biological effects of low and high dose radiation and individual variation in radiosensitivity. Most of the data on the effect of radiation have been accumulated from atomic bomb survivors, the only large cohort of exposed individuals that has been carefully monitored. Genetic study of radiosensitivity is difficult because relatives rarely get exposed to the same type and doses of radiation. In this project, we will use a combined genomic and genetic approach to determine the mechanisms of cellular response to different doses of radiation and to identify genetic variants that influence radiosensitivity. We will use expression level of genes in irradiated cells as phenotypes. This allows us to expose cells to different doses of radiation and to obtain radiation-induced phenotypes from related individuals for genetic analysis. The specific aims are 1) determine and compare the gene expression phenotypes induced by 0.5 Gy and 3 Gy IR, 2) map the chromosomal regions that influence inherited variation in IR responsive genes in large families by linkage analysis, and 3) confirm the linkage results and narrow the candidate gene regions by association analysis, and functionally characterize candidate regulators of response to 0.5 Gy and 3 Gy IR exposures. The results will provide information on the molecular and genetic basis of individual response to radiation exposure and form a foundation for a personalized approach to risk prediction.

The focus of this study is the genetics of variation in human gene expression. Our overall goals are to characterize the extent of variation in gene expression and to identify the genetic determinants of this variation. The specific aims for this renewal application are: Aim 1. Expand materials and test for replication of linkage/association in an independent sample of families. Aim 2. Carry out family studies of differential allelic expression. Aim 3. Characterize the transcriptional regulatory regions. In the first two years of the current three-year grant, we have determined the gene expression phenotypes of members of approximately40 large families and carried out linkage analysis to determine the chromosomal location linked to each phenotype. The findings were followed up by genome-wide association analysis of the expression phenotypes, using SNP genotypes in samples from the International HapMap Project. In this renewal application, we will extend our genetic study to 45 additional families. The new phenotype data, along with SNP genotypes of the same individuals, will be used to evaluate replication of findings from the original genome-wide linkage and association analyses, and to strengthen the evidence for positive results. To complement our findings of differential allelic expression from linkage and association, we will carry out analysis of "allelic imbalance" in monozygotic twins and family members. By measuring the expression of transcripts from the two alleles of a gene, we get a direct assessment of cis-acting regulatory effects on gene expression. Results from such analyses have revealed extensive variability in the nature and extent of allelic imbalance. Our family-based approach will allow us to assess the relative contributions of inherited cis and trans regulators, and of imprinting, to this variability. Once we have identified candidate regions that contain cis- and/ or trans-acting transcriptional regulators, we will perform molecular characterization of those regions in order to identify the sequence variants responsible for the observed variation in gene expression, and determine the regulatory mechanisms. Gene expression is the link between DNA sequence and phenotype variation, including disease. Our approach will allow us to characterize gene expression variation in humans and to understand transcriptional control by identifying transcriptional regulators. The level of gene expression is also a paradigm for other quantitative traits. Therefore, the molecular and analytical approaches developed here can be generalized and applied to the study of other quantitative traits in humans, including complex genetic diseases.

DESCRIPTION (provided by applicant): Regulatory variants play important roles in disease susceptibility. Results from genome-wide association have identified many DNA variants that are associated with diseases. However, how these variants influence disease susceptibility is largely unknown. Some of these variants regulate gene expression but the target genes of these regulatory variants have yet to be identified. A challenge is to decide the relevant cell types/ tissues for functional analyses. In human studies, many cell types are not readily available for experimentation. Thus, the knowledge of cell type specificities in gene regulation is important. If many genes are widely expressed and their regulations are similar across multiple cell types, then one can use readily available tissues for functional studies, and expect the results to generalize. In this R21 project, our goal is to determine the number (and identity) of genes that are widely expressed in the human genome, and the extent to which their expression is shared across cell types. We will also examine whether regulatory variants of widely expressed genes are susceptibility variants for human traits and diseases. The specific aims are: 1) identify genes that are expressed across multiple tissues and conditions by in silico analysis of gene expression data in public repositories and deep sequencing, 2) compare regulation of genes that are expressed across different human cells by network analysis and gene mapping studies, and 3) determine whether regulatory variants identified in Aim 2 are susceptibility alleles for common complex diseases. The findings will guide functional studies to identify the regulatory roles of disease susceptibility variants. The results will also provide examples of disease susceptibility alleles that influence expression levels of human genes. Numerous studies have identified DNA sequences that influence a person's risk of developing diseases. An important next step is to study how these DNA sequences affect disease development. Human studies are difficult because many cell types are not available for experimental manipulations. Our study is designed to establish whether regulatory functions are shared among different cells. The results will help to determine whether easily accessible cells such as blood and skin cells can be used as proxies to examine the biological functions of DNA sequences that influence disease risks.