Raju S Kucherlapati - US grants

DESCRIPTION (Applicant's Abstract): The goal of this project is to contribute to the determination of the accurate sequence of human chromosome 12. Towards this goal, we entered into a collaboration with the Baylor College of Medicine (BCM) Genome Center. We propose to generate a sequence ready map of chromosome 12 in the form of P1 artificial chromosome (PAC) and bacterial artificial chromosome (BAC) maps. During the first year of the proposed grant, we propose to construct a map for 20 Mbp of chromosome 12, implement a bioinformatics strategy and put in place a number of high throughput systems to reach a production level mapping. During years two and three, we propose to construct maps for 100 Mbp of chromosome 12 and provide them to the BCM Genome Center. Based on this throughput, the sequence ready maps of chromosome 12 will be completed by December 31, 2000 and the sequence will be completed shortly thereafter. The general approach to construct the maps is as follows. We will use the 100 Kbp resolution physical map of human chromosome 12 in the form of a YAC contig map that we constructed as the framework map. PCR products from ten to twelve consecutive non-polymorphic markers will be used to screen a 20X genomic PAC/BAC library. False positive will be eliminated by a PCR based screening assay and the clones will be assembled into contigs by STS-content. Marker density will be increased by sequencing ends of clones by a direct sequencing method we developed and developing new STSs. Gaps will be filled by screening libraries with end clones. Clones that can constitute minimal tiling paths will be identified by fingerprinting methods. All of the data and the minimal tiling path of clones will be electronically transmitted to the BCM Genome Center and made publicly available through a web site. A team of six mappers assisted by an Informatics team will produce 20 Mbp of maps during the first year. We will implement automated methods for several steps in the procedure and, during the second and third years, we propose to increase the throughput by 2X with only the addition of two FTEs to the overall team. During years 2 and 3, the cost of producing the maps is estimated to be no more than $.025/nucleotide.

DESCRIPTION (Taken from the Applicant?s Abstract) The applicants propose to become members of the Comparative Mouse Genomics Centers Consortium. A diverse group of investigators from four different institutions will participate in this program. The Kolodner laboratory will identify novel genes involved in DNA repair and DNA replication and examine the human population for polymorphisms that have functional significance. The functional importance of the polymorphisms will be assessed by the Kolodner and Kunkel laboratories by using yeast-based assays. The Edelmann and Kucherlapati laboratories will use modern gene modification technologies to generate mice that have null and specific point mutations in genes of interest. The Kunkel laboratory will characterize the biochemical properties of cells obtained from the mutant mice, and the Wang laboratory will examine the effects of DNA damaging agents on these mice. The Russell and Lipkin laboratories will examine the pathological consequences of the mutations under normal and different dietary regimens. The participating laboratories have experience in molecular biology, cell biology, and pathology. In addition, this group will bring several new technologies for the Consortium. These include high throughput physical mapping of segments of the mouse genome, high throughput DNA sequencing, and genetic analysis methods. The Kucherlapati laboratory will also bring expertise in the use and analysis of DNA microarray technologies including cDNA and oligonucleotide array methods. They also propose to develop tissue arrays. Experience in bioinformatics will be used to disseminate the data generated by this group. The investigators also propose to develop and implement novel technologies that might be of value to all members of the consortium.

DESCRIPTION (provided by applicant): Velo-cardio-facial syndrome and DiGeorge syndrome are relatively common disorders and affect at least 1/4000 newborn children. Most of the cases of VCFS/DGS are sporadic. Children with these syndromes present with a number of abnormalities including facial dysmorphology, cardiovascular defects, immune defects, speech defects and learning disabilities. As the children grow older, many of them develop severe psychiatric illness. A subset of the patients diagnosed to have VCFS/DGS have a deletion of either 1.5 or 3.0 Mb of DNA on human chromosome 22ql 1. The phenotypes associated with VCFS/DGS are quite variable. Individuals within the same family who carry the same deletion sometimes have different phenotypic manifestations. Because of the variability in the phenotypic spectrum and since not all cases may result from abnormalities in chromosome 22 there is a strong need to develop quantitative clinical criteria for VCFS/DGS diagnosis. It is also possible that a careful clinical data acquisition would allow us to classify and sub classify the patient population. Many of the organs systems affected in VCFS/DGS patients have their origin in the neural crest. This includes the face. Although it is well recognized that facial dysmorphology is an important component of VCFS/DGS phenotype no good quantitative criteria for describing the facial dysmorphology are available. We propose to use an emerging method of three-dimensional photogrammetry to collect information about the facial features of patients clinically diagnosed to have VCFS/DGS. This method is rapid, analogous to taking a photograph, and provides a digitized three-dimensional image of the face of an individual. Software programs written by our colleagues and applied by us allow a large number of measurements that are accurate and reproducible. During the three-year period, we will enroll 300 patients in the study and obtain photogrammetric measurements as well as other clinical parameters from these patients. We hypothesize that facial features can be used as accurate diagnostic measures of these syndromes and that they would correlate with the deletion status and the size of the Ideletion. We have assembled a group of clinical and basic scientists as well a bioinformatician to address this problem.

DESCRIPTION (provided by applicant): Velo cardio facial syndrome and DiGeorge syndrome (VCFS/DGS) are the most common human developmental disorders that result from haploinsufficiency. Most of the patients are hemizygous for 3 Mb deletion on human chromosome 22q11. Patients with VCFS/DGS have a number of abnormalities in tissues and organ systems that are derived from the neural crest. These include facial dysmorphology, cardiovascular defects, immune system defects and many others. Using the mouse as a model system the PIs have been able to show that mice that either overexpress or have reduced expression of Tbx1, a gene located in the deleted interval, have defects in many of the organs systems affected in VCFS/DGS patients. TBX1 encodes a transcription factor that is critical for normal neural crest development. Although information about the mechanisms of action of TBX1 is emerging, no systematic efforts to identify the pathways and networks in which TBX1 acts have been undertaken. The PIs propose to do so. TBX1 is highly conserved during evolution and mutations of this gene in the zebrafish, designated van gogh (vgo), develop phenotypes that are similar to those seen in VCFS/DGS patients. Based on these results, the PIs now propose a comparative genetics analysis of the TBX1 pathways and networks in zebrafish and mice. They propose to use gene expression profiling of normal and mutant zebrafish and mouse embryos at the appropriate developmental stages to discover genes whose expression patterns are altered as a result of reduction or absence of TBX1 protein. They will use whole mount in situ hybridization methods to examine the spatiotemporal patterns of expression of a large number of these genes in zebrafish. A subset will also be examined in Tbx1 mutant zebrafish and mice. Genes whose expression patterns match that of TBX1 or in appropriate cells and tissues will be subjected to genetic analysis in zebrafish through overexpression and morpholino-based knock-downs. If mutants for some of these genes are available, they will be examined alone and in combination with TBX1 mutants. In some cases the PIs will generate null and conditional mutant alleles in mice to examine their role in development. The combination of the use of zebrafish and mouse together with high throughput genetic and genomic approaches promise to provide rich information about the pathways and networks in which TBX1 operates.

DESCRIPTION (provided by applicant): We are seeking support to establish a Genome Characterization Center, as a part of the Cancer Genome Atlas Project in Boston. The goal of the proposed effort is to analyze 2,000-2,500 tumor samples each year over a five-year period of time and identify a set of genes that can be resequenced by the members of The Cancer Genome Atlas (TCGA) project. It is well established that regions of the cancer genome that are amplified or show loss of heterozygosity or deletion harbor genes that are important for tumor initiation and progression. We will initially identify such regions in the cancer genome by conducting array comparative genomic hybridization (aCGH). Based upon detailed comparisons of many different platforms we have chosen to use the high-density Agilent oligonucleotide arrays for our studies. We have used the Agilent platform to characterize several hundreds of tumors and their corresponding controls, much of it a part of the current TCGA project. We already have the ability to have a throughput of processing four to five thousand samples during the first year of the proposed grant. During the first year we propose to use a sequencing based approach to determine copy number changes in tumors. In the initial phase we will examine 200 samples by a sequence tag counting approach and compare the results with the Agilent and other platforms. Based upon our experience we anticipate that this is eminently feasible and we will gradually switch the copy number analysis to the sequence based platform. In the second year 40% of the samples will be processed by the sequencing platform and the remaining 60% by the array platform. In year three 60% of the samples will be processed by sequencing and in years 4 and 5 we plan to completely switch to the sequencing approach. We have a well established pipeline to collect, store, retrieve and analyze the data from these two platforms. We will develop Level 1-4 data as defined by the consortium and deposit these data in a timely fashion in the Data Coordinating Center. We also propose to use powerful informatics tools that we have developed and propose to improve to analyze the aCGH and the sequence based data and extract a list of most interesting genes for resequencing. We have established an award winning IT infrastructure that will be deployed for LIMS, data storage, data retrieval, data analysis and interface with caBIG. Our proposed approach also has the ability to generate additional useful data for tumor and patient stratification.