Project description:Clinical DNA is often available in limited quantities requiring whole-genome amplification for subsequent genome-wide assessment of copy-number variation (CNV) by array-CGH. In pre-implantation diagnosis and analysis of micrometastases, even merely single cells are available for analysis. However, procedures allowing high-resolution analyses of CNVs from single cells well below resolution limits of conventional cytogenetics are lacking. Here, we applied amplification products of single cells and of cell pools (5 or 10 cells) from patients with developmental delay, cancer cell lines and polar bodies to various oligo tiling array platforms with a median probe spacing as high as 65 bp. Our high-resolution analyses reveal that the low amounts of template DNA do not result in a completely unbiased whole genome amplification but that stochastic amplification artifacts, which become more obvious on array platforms with tiling path resolution, cause significant noise. We implemented a new evaluation algorithm specifically for the identification of small gains and losses in such very noisy ratio profiles. Our data suggest that when assessed with sufficiently sensitive methods high-resolution oligo-arrays allow a reliable identification of CNVs as small as 500 kb in cell pools (5 or 10 cells), and of 2.6-3.0 Mb in single cells.
Project description:Kynureninase is a member of a large family of catalytically diverse but structurally homologous pyridoxal 5'-phosphate (PLP) dependent enzymes known as the aspartate aminotransferase superfamily or alpha-family. The Homo sapiens and other eukaryotic constitutive kynureninases preferentially catalyze the hydrolytic cleavage of 3-hydroxy-l-kynurenine to produce 3-hydroxyanthranilate and l-alanine, while l-kynurenine is the substrate of many prokaryotic inducible kynureninases. The human enzyme was cloned with an N-terminal hexahistidine tag, expressed, and purified from a bacterial expression system using Ni metal ion affinity chromatography. Kinetic characterization of the recombinant enzyme reveals classic Michaelis-Menten behavior, with a Km of 28.3 +/- 1.9 microM and a specific activity of 1.75 micromol min-1 mg-1 for 3-hydroxy-dl-kynurenine. Crystals of recombinant kynureninase that diffracted to 2.0 A were obtained, and the atomic structure of the PLP-bound holoenzyme was determined by molecular replacement using the Pseudomonas fluorescens kynureninase structure (PDB entry 1qz9) as the phasing model. A structural superposition with the P. fluorescens kynureninase revealed that these two structures resemble the "open" and "closed" conformations of aspartate aminotransferase. The comparison illustrates the dynamic nature of these proteins' small domains and reveals a role for Arg-434 similar to its role in other AAT alpha-family members. Docking of 3-hydroxy-l-kynurenine into the human kynureninase active site suggests that Asn-333 and His-102 are involved in substrate binding and molecular discrimination between inducible and constitutive kynureninase substrates.
Project description:Breast cancer remains the second leading cause of cancer-related death in women despite stratification based on standard hormonal receptor (HR) and HER2 testing. Additional prognostic markers are needed to improve breast cancer treatment. Chromothripsis, a catastrophic genome rearrangement, has been described recently in various cancer genomes and affects cancer progression and prognosis. However, little is known about chromothripsis in breast cancer. To identify novel prognostic biomarkers in breast cancer, we used molecular inversion probe (MIP) microarray to explore genome-wide copy number aberrations (CNA) and breast cancer-related gene alterations in DNA extracted from formalin-fixed paraffin-embedded tissue. We examined 42 primary breast cancers with known HR and HER2 status assessed via immunohistochemistry and FISH and analyzed MIP microarray results for correlation with standard tests and survival outcomes. Global genome-wide CNA ranged from 0.2% to 65.7%. Chromothripsis-like patterns were observed in 23/38 (61%) cases and were more prevalent in cases with ?10% CNA (20/26, 77%) than in cases with <10% CNA (3/12, 25%; p<0.01). Most frequently involved chromosomal segment was 17q12-q21, the HER2 locus. Chromothripsis-like patterns involving 17q12 were observed in 8/19 (42%) of HER2-amplified tumors but not in any of the tumors without HER2 amplification (0/19; p<0.01). HER2 amplification detected by MIP microarray was 95% concordant with conventional testing (39/41). Interestingly, 21% of patients (9/42) had fibroblast growth factor receptor 1 (FGFR1)amplification and had a 460% higher risk for mortality than those without FGFR1 amplification (p<0.01). In summary, MIP microarray provided a robust assessment of genomic CNA of breast cancer.
Project description:By using the 1.6 million single-nucleotide polymorphism (SNP) genotype data set from Perlegen Sciences [Hinds, D. A., Stuve, L. L., Nilsen, G. B., Halperin, E., Eskin, E., Ballinger, D. G., Frazer, K. A. & Cox, D. R. (2005) Science 307, 1072-1079], a probabilistic search for the landscape exhibited by positive Darwinian selection was conducted. By sorting each high-frequency allele by homozygosity, we search for the expected decay of adjacent SNP linkage disequilibrium (LD) at recently selected alleles, eliminating the need for inferring haplotype. We designate this approach the LD decay (LDD) test. By these criteria, 1.6% of Perlegen SNPs were found to exhibit the genetic architecture of selection. These results were confirmed on an independently generated data set of 1.0 million SNP genotypes (International Human Haplotype Map Phase I freeze). Simulation studies indicate that the LDD test, at the megabase scale used, effectively distinguishes selection from other causes of extensive LD, such as inversions, population bottlenecks, and admixture. The approximately 1,800 genes identified by the LDD test were clustered according to Gene Ontology (GO) categories. Based on overrepresentation analysis, several predominant biological themes are common in these selected alleles, including host-pathogen interactions, reproduction, DNA metabolism/cell cycle, protein metabolism, and neuronal function.
Project description:Understanding factors required for DNA replication will enrich our knowledge of this important process and potentially identify vulnerabilities that can be exploited in cancer therapy. We applied an assay that measures the stability of maintenance of an episomal plasmid in human tissue culture cells to screen for new DNA replication factors. We identify an important role for DDX5 in G(1)-S-phase progression where it directly regulates DNA replication factor expression by promoting the recruitment of RNA polymerase II to E2F-regulated gene promoters. We find that the DDX5 locus is frequently amplified in breast cancer and that breast cancer-derived cells with amplification of DDX5 are much more sensitive to its depletion than breast cancer cells and a breast epithelial cell line that lacks DDX5 amplification. Our results show a novel role for DDX5 in cancer cell proliferation and suggest DDX5 as a therapeutic target in breast cancer treatment.DDX5 is required for cell proliferation by controlling the transcription of genes expressing DNA replication proteins in cancer cells in which the DDX5 locus is amplified, and this has uncovered a dependence on DDX5 for cell proliferation. Given the high frequency of DDX5 amplification in breast cancer, our results highlight DDX5 as a promising candidate for targeted therapy of breast tumors with DDX5 amplification, and indeed we show that DDX5 inhibition sensitizes a subset of breast cancer cells to trastuzumab.
Project description:We investigated the evidence of recent positive selection in the human phototransduction system at single nucleotide polymorphism (SNP) and gene level.SNP genotyping data from the International HapMap Project for European, Eastern Asian, and African populations was used to discover differences in haplotype length and allele frequency between these populations. Numeric selection metrics were computed for each SNP and aggregated into gene-level metrics to measure evidence of recent positive selection. The level of recent positive selection in phototransduction genes was evaluated and compared to a set of genes shown previously to be under recent selection, and a set of highly conserved genes as positive and negative controls, respectively.Six of 20 phototransduction genes evaluated had gene-level selection metrics above the 90th percentile: RGS9, GNB1, RHO, PDE6G, GNAT1, and SLC24A1. The selection signal across these genes was found to be of similar magnitude to the positive control genes and much greater than the negative control genes.There is evidence for selective pressure in the genes involved in retinal phototransduction, and traces of this selective pressure can be demonstrated using SNP-level and gene-level metrics of allelic variation. We hypothesize that the selective pressure on these genes was related to their role in low light vision and retinal adaptation to ambient light changes. Uncovering the underlying genetics of evolutionary adaptations in phototransduction not only allows greater understanding of vision and visual diseases, but also the development of patient-specific diagnostic and intervention strategies.
Project description:Amplification of the HER-2/neu (HER-2) proto-oncogene occurs in 10%-15% of primary breast cancer, leading to an activated HER-2 receptor, augmenting growth of cancer cells. Tumor classification is determined in primary tumor tissue and metastatic biopsies. However, malignant cells tend to alter their phenotype during disease progression. Circulating tumor cell (CTC) analysis may serve as an alternative to repeated biopsies. The Food and Drug Administration-approved CellSearch system allows determination of the HER-2 protein, but not of the HER-2 gene. The aim of this study was to optimize a fluorescence in situ hybridization (FISH)-based method to quantitatively determine HER-2 amplification in breast cancer CTCs following CellSearch-based isolation and verify the method in patient samples.Using healthy donor blood spiked with human epidermal growth factor receptor 2 (HER-2)-positive breast cancer cell lines, SKBr-3 and BT-474, and a corresponding negative control (the HER-2-negative MCF-7 cell line), an in vitro CTC model system was designed. Following isolation in the CellSearch system, CTC samples were further enriched and fixed on microscope slides. Immunocytochemical staining with cytokeratin and 4',6-diamidino-2'-phenylindole dihydrochloride identified CTCs under a fluorescence microscope. A FISH-based procedure was optimized by applying the HER2 IQFISH pharmDx assay for assessment of HER-2 amplification status in breast cancer CTCs.A method for defining the presence of HER-2 amplification in single breast cancer CTCs after CellSearch isolation was established using cell lines as positive and negative controls. The method was validated in blood from breast cancer patients showing that one out of six patients acquired CTC HER-2 amplification during treatment against metastatic disease.HER-2 amplification status of CTCs can be determined following CellSearch isolation and further enrichment. FISH is superior to protein assessment of HER-2 status in predicting response to HER-2-targeted immunotherapy in breast cancer patients. This assay has the potential of identifying patients with a shift in HER-2 status who may benefit from treatment adjustments.