Biomedical subjects
Ronald W Davis
Publications and source records attributed to Ronald W Davis.
Single cell mutational analysis of PIK3CA in circulating tumor cells and metastases in breast cancer reveals heterogeneity, discordance, and mutation persistence in cultured disseminated tumor cells from bone marrow.
BACKGROUND: Therapeutic decisions in cancer are generally guided by molecular biomarkers or, for some newer therapeutics, primary tumor genotype. However, because biomarkers or genotypes may change as new metastases emerge, circulating tumor cells (CTCs) from blood are being investigated for a role in guiding real-time drug selection during disease progression, expecting that CTCs will comprehensively represent the full spectrum of genomic changes in metastases. However, information is limited regarding mutational heterogeneity among CTCs and metastases in breast cancer as discerned by single cell analysis. The presence of disseminated tumor cells (DTCs) in bone marrow also carry prognostic significance in breast cancer, but with variability between CTC and DTC detection. Here we analyze a series of single tumor cells, CTCs, and DTCs for PIK3CA mutations and report CTC and corresponding metastatic genotypes. METHODS: We used the MagSweeper, an immunomagnetic separation device, to capture live single tumor cells from breast cancer patients' primary and metastatic tissues, blood, and bone marrow. Single cells were screened for mutations in exons 9 and 20 of the PIK3CA gene. Captured DTCs grown in cell culture were also sequenced for PIK3CA mutations. RESULTS: Among 242 individual tumor cells isolated from 17 patients and tested for mutations, 48 mutated tumor cells were identified in three patients. Single cell analyses revealed mutational heterogeneity among CTCs and tumor cells in tissues. In a patient followed serially, there was mutational discordance between CTCs, DTCs, and metastases, and among CTCs isolated at different time points. DTCs from this patient propagated in vitro contained a PIK3CA mutation, which was maintained despite morphological changes during 21 days of cell culture. CONCLUSIONS: Single cell analysis of CTCs can demonstrate genotypic heterogeneity, changes over time, and discordance from DTCs and distant metastases. We present a cautionary case showing that CTCs from any single blood draw do not always reflect metastatic genotype, and that CTC and DTC analyses may provide independent clinical information. Isolated DTCs remain viable and can be propagated in culture while maintaining their original mutational status, potentially serving as a future resource for investigating new drug therapies.
Contributions of ATM mutations to familial breast and ovarian cancer.
This study addresses the prevalence of ATM mutations and the association with breast cancer in Austrian families selected for a history of breast or ovarian cancer or both [hereditary breast and ovarian cancer (HBOC)]. In 270 HBOC families previously screened for BRCA1 and BRCA2 mutations, 137 different sequence alterations of ATM were identified. Seven of these were mutations presumed to cause ataxia telangiectasia based on their effect on the ATM protein, including five that caused a protein truncation and two missense mutations in the catalytic kinase domain of the highly conserved COOH terminus of the protein. The seven mutations were found in 10 families (3.7%). In addition, one missense variant, L1420F, was observed in 13 HBOC families (4.8%) but was not observed in any of the 122 healthy volunteers with no history of breast cancer. In addition, the variant segregated with breast cancer in some of the families, suggesting that it may be pathogenic for breast cancer. Sixty-two additional variants of potential significance were observed in 65 HBOC families, but not in healthy controls. These variants included 24 sequence alterations with possible effects on splicing or protein-protein interactions. This study indicates that there is a significant prevalence of ATM mutations in breast and ovarian cancer families and adds to a growing body of evidence that ATM mutations confer increased susceptibility to breast cancer.
Multiplexed genotyping with sequence-tagged molecular inversion probes.
We report on the development of molecular inversion probe (MIP) genotyping, an efficient technology for large-scale single nucleotide polymorphism (SNP) analysis. This technique uses MIPs to produce inverted sequences, which undergo a unimolecular rearrangement and are then amplified by PCR using common primers and analyzed using universal sequence tag DNA microarrays, resulting in highly specific genotyping. With this technology, multiplex analysis of more than 1,000 probes in a single tube can be done using standard laboratory equipment. Genotypes are generated with a high call rate (95%) and high accuracy (>99%) as determined by independent sequencing.
Role of duplicate genes in genetic robustness against null mutations.
Deleting a gene in an organism often has little phenotypic effect, owing to two mechanisms of compensation. The first is the existence of duplicate genes: that is, the loss of function in one copy can be compensated by the other copy or copies. The second mechanism of compensation stems from alternative metabolic pathways, regulatory networks, and so on. The relative importance of the two mechanisms has not been investigated except for a limited study, which suggested that the role of duplicate genes in compensation is negligible. The availability of fitness data for a nearly complete set of single-gene-deletion mutants of the Saccharomyces cerevisiae genome has enabled us to carry out a genome-wide evaluation of the role of duplicate genes in genetic robustness against null mutations. Here we show that there is a significantly higher probability of functional compensation for a duplicate gene than for a singleton, a high correlation between the frequency of compensation and the sequence similarity of two duplicates, and a higher probability of a severe fitness effect when the duplicate copy that is more highly expressed is deleted. We estimate that in S. cerevisiae at least a quarter of those gene deletions that have no phenotype are compensated by duplicate genes.
Determination of hepatitis C virus genotype by Pyrosequencing.
A simple sequencing-based assay is described for genotyping of hepatitis C virus (HCV). RT-PCR was employed to amplify a 237-nucleotide-long fragment from the 5' untranslated region (UTR) of the genome using one biotinylated and one normal primer. Subsequent to capture of the PCR products on streptavidin-coated beads, single-stranded DNA separation, and hybridization of sequencing primer, Pyrosequencing was performed. The genotype of 98 samples out of which 77 samples were from American veterans and 21 samples were from Iran was determined. The samples from the American veterans contained six different subtypes, while five subtypes were found in Iranian samples. For rapid population-specific HCV subtyping, a multiplex assay was developed. This study demonstrates the suitability of this technology for low-cost, high throughput and accurate microbial genotyping.
Parallel phenotypic analysis of sporulation and postgermination growth in Saccharomyces cerevisiae.
We have quantitatively monitored the sporulation and germination efficiencies of approximately 4,200 yeast deletion strains in parallel by using a molecular bar coding strategy. In a single study, we doubled the number of genes functionally implicated in sporulation to approximately 400, identifying both positive and negative regulators. Our set of 261 sporulation-deficient genes illustrates the importance of autophagy, carbon utilization, and transcriptional machinery during sporulation. These general cellular factors are more likely to exhibit fitness defects when deleted and less likely to be transcriptionally regulated than sporulation-specific genes. Our postgermination screening assay identified recombinationchromosome segregation genes, aneuploid strains, and possible germination-specific factors. Finally, our results facilitate a genome-wide comparison of expression pattern and mutant phenotype for a developmental process and suggest that 16% of genes differentially expressed during sporulation confer altered efficiency of spore production or defective postgermination growth when disrupted.
Metabolic specialization associated with phenotypic switching in Candidaalbicans.
Phase and antigenic variation are mechanisms used by microbial pathogens to stochastically change their cell surface composition. A related property, referred to as phenotypic switching, has been described for some pathogenic fungi. This phenomenon is best studied in Candida albicans, where switch phenotypes vary in morphology, physiology, and pathogenicity in experimental models. In this study, we report an application of a custom Affymetrix GeneChip representative of the entire C. albicans genome and assay the global expression profiles of white and opaque switch phenotypes of the WO-1 strain. Of 13,025 probe sets examined, 373 ORFs demonstrated a greater than twofold difference in expression level between switch phenotypes. Among these, 221 were expressed at a level higher in opaque cells than in white cells; conversely, 152 were more highly expressed in white cells. Affected genes represent functions as diverse as metabolism, adhesion, cell surface composition, stress response, signaling, mating type, and virulence. Approximately one-third of the differences between cell types are related to metabolic pathways, opaque cells expressing a transcriptional profile consistent with oxidative metabolism and white cells expressing a fermentative one. This bias was obtained regardless of carbon source, suggesting a connection between phenotypic switching and metabolic flexibility, where metabolic specialization of switch phenotypes enhances selection in relation to the nutrients available at different anatomical sites. These results extend our understanding of strategies used in microbial phase variation and pathogenesis and further characterize the unanticipated diversity of genes expressed in phenotypic switching.
The Ume6 regulon coordinates metabolic and meiotic gene expression in yeast.
The Ume6 transcription factor in yeast is known to both repress and activate expression of diverse genes during growth and meiotic development. To obtain a more complete profile of the functions regulated by this protein, microarray analysis was used to examine transcription in wild-type and ume6Delta diploids during vegetative growth in glucose and acetate. Two different genetic backgrounds (W303 and SK1) were examined to identify a core set of strain-independent Ume6-regulated genes. Among genes whose expression is controlled by Ume6 in both backgrounds, 82 contain homologies to the Ume6-binding site (URS1) and are expected to be directly regulated by Ume6. The vast majority of those whose functions are known participate in carbon/nitrogen metabolism and/or meiosis. Approximately half of the Ume6 direct targets are induced during meiosis, with most falling into the early meiotic expression class (cluster 4), and a smaller subset in the middle and later classes (clusters 5-7). Based on these data, we propose that Ume6 serves a unique role in diploid cells, coupling metabolic responses to nutritional cues with the initiation and progression of meiosis. Finally, expression patterns in the two genetic backgrounds suggest that SK1 is better adapted to respiration and W303 to fermentation, which may in part account for the more efficient and synchronous sporulation of SK1.
Genome sequence of the human malaria parasite Plasmodium falciparum.
The parasite Plasmodium falciparum is responsible for hundreds of millions of cases of malaria, and kills more than one million African children annually. Here we report an analysis of the genome sequence of P. falciparum clone 3D7. The 23-megabase nuclear genome consists of 14 chromosomes, encodes about 5,300 genes, and is the most (A + T)-rich genome sequenced to date. Genes involved in antigenic variation are concentrated in the subtelomeric regions of the chromosomes. Compared to the genomes of free-living eukaryotic microbes, the genome of this intracellular parasite encodes fewer enzymes and transporters, but a large proportion of genes are devoted to immune evasion and host-parasite interactions. Many nuclear-encoded proteins are targeted to the apicoplast, an organelle involved in fatty-acid and isoprenoid metabolism. The genome sequence provides the foundation for future studies of this organism, and is being exploited in the search for new drugs and vaccines to fight malaria.
Sequence of Plasmodium falciparum chromosome 12.
The human malaria parasite Plasmodium falciparum is responsible for the death of more than a million people every year. To stimulate basic research on the disease, and to promote the development of effective drugs and vaccines against the parasite, the complete genome of P. falciparum clone 3D7 has been sequenced, using a chromosome-by-chromosome shotgun strategy. Here we report the nucleotide sequence of the third largest of the parasite's 14 chromosomes, chromosome 12, which comprises about 10% of the 23-megabase genome. As the most (A + T)-rich (80.6%) genome sequenced to date, the P. falciparum genome presented severe problems during the assembly of primary sequence reads. We discuss the methodology that yielded a finished and fully contiguous sequence for chromosome 12. The biological implications of the sequence data are more thoroughly discussed in an accompanying Article (ref. 3).
Gene expression during the life cycle of Drosophila melanogaster.
Molecular genetic studies of Drosophila melanogaster have led to profound advances in understanding the regulation of development. Here we report gene expression patterns for nearly one-third of all Drosophila genes during a complete time course of development. Mutations that eliminate eye or germline tissue were used to further analyze tissue-specific gene expression programs. These studies define major characteristics of the transcriptional programs that underlie the life cycle, compare development in males and females, and show that large-scale gene expression data collected from whole animals can be used to identify genes expressed in particular tissues and organs or genes involved in specific biological and biochemical processes.
Functional profiling of the Saccharomyces cerevisiae genome.
Determining the effect of gene deletion is a fundamental approach to understanding gene function. Conventional genetic screens exhibit biases, and genes contributing to a phenotype are often missed. We systematically constructed a nearly complete collection of gene-deletion mutants (96% of annotated open reading frames, or ORFs) of the yeast Saccharomyces cerevisiae. DNA sequences dubbed 'molecular bar codes' uniquely identify each strain, enabling their growth to be analysed in parallel and the fitness contribution of each gene to be quantitatively assessed by hybridization to high-density oligonucleotide arrays. We show that previously known and new genes are necessary for optimal growth under six well-studied conditions: high salt, sorbitol, galactose, pH 8, minimal medium and nystatin treatment. Less than 7% of genes that exhibit a significant increase in messenger RNA expression are also required for optimal growth in four of the tested conditions. Our results validate the yeast gene-deletion collection as a valuable resource for functional genomics.
Systematic screen for human disease genes in yeast.
High similarity between yeast and human mitochondria allows functional genomic study of Saccharomyces cerevisiae to be used to identify human genes involved in disease. So far, 102 heritable disorders have been attributed to defects in a quarter of the known nuclear-encoded mitochondrial proteins in humans. Many mitochondrial diseases remain unexplained, however, in part because only 40-60% of the presumed 700-1,000 proteins involved in mitochondrial function and biogenesis have been identified. Here we apply a systematic functional screen using the pre-existing whole-genome pool of yeast deletion mutants to identify mitochondrial proteins. Three million measurements of strain fitness identified 466 genes whose deletions impaired mitochondrial respiration, of which 265 were new. Our approach gave higher selection than other systematic approaches, including fivefold greater selection than gene expression analysis. To apply these advantages to human disorders involving mitochondria, human orthologs were identified and linked to heritable diseases using genomic map positions.
Transcriptional response of Saccharomyces cerevisiae to DNA-damaging agents does not identify the genes that protect against these agents.
The recent completion of the deletion of all of the nonessential genes in budding yeast has provided a powerful new way of determining those genes that affect the sensitivity of this organism to cytotoxic agents. We have used this system to test the hypothesis that genes whose transcription is increased after DNA damage are important for the survival to that damage. We used a pool of 4,627 diploid strains each with homozygous deletion of a nonessential gene to identify those genes that are important for the survival of yeast to four DNA-damaging agents: ionizing radiation, UV radiation, and exposure to cisplatin or to hydrogen peroxide. In addition we measured the transcriptional response of the wild-type parental strain to the same DNA-damaging agents. We found no relationship between the genes necessary for survival to the DNA-damaging agents and those genes whose transcription is increased after exposure. These data show that few, if any, of the genes involved in repairing the DNA lesions produced in this study, including double-strand breaks, pyrimidine dimers, single-strand breaks, base damage, and DNA cross-links, are induced in response to toxic doses of the agents that produce these lesions. This finding suggests that the enzymes necessary for the repair of these lesions are at sufficient levels within the cell. The data also suggest that the nature of the lesions produced by DNA-damaging agents cannot easily be deduced from gene expression profiling.
Multiplex Pyrosequencing.
We describe here the development of a new and simple single-tube multiplex Pyrosequencing assay. Genomic DNA or cDNA was employed to PCR amplify region(s) using biotinylated and normal primer(s). Subsequent to capture of PCR products on streptavidin-coated beads, single-stranded DNA separation and hybridization of multiple sequencing primers, Pyrosequencing was performed. The obtained pyrogram resulted in a unique pattern in which the intensity of the signal determined the number of incorporated nucleotide(s). Here, we demonstrate the use of this multiplex Pyrosequencing for single nucleotide polymorphisms genotyping and microbial typing.
Dissecting the architecture of a quantitative trait locus in yeast.
Most phenotypic diversity in natural populations is characterized by differences in degree rather than in kind. Identification of the actual genes underlying these quantitative traits has proved difficult. As a result, little is known about their genetic architecture. The failures are thought to be due to the different contributions of many underlying genes to the phenotype and the ability of different combinations of genes and environmental factors to produce similar phenotypes. This study combined genome-wide mapping and a new genetic technique named reciprocal-hemizygosity analysis to achieve the complete dissection of a quantitative trait locus (QTL) in Saccharomyces cerevisiae. A QTL architecture was uncovered that was more complex than expected. Functional linkages both in cis and in trans were found between three tightly linked quantitative trait genes that are neither necessary nor sufficient in isolation. This arrangement of alleles explains heterosis (hybrid vigour), the increased fitness of the heterozygote compared with homozygotes. It also demonstrates a deficiency in current approaches to QTL dissection with implications extending to traits in other organisms, including human genetic diseases.
Identification and preliminary characterization of mouse Adam33.
BACKGROUND: The metalloprotease-disintegrin family, or ADAM, proteins, are implicated in cell-cell interactions, cell fusion, and cell signaling, and are widely distributed among metazoan phyla. Orthologous relationships have been defined for a few ADAM proteins including ADAM10 (Kuzbanian), and ADAM17 (TACE), but evolutionary relationships are not clear for the majority of family members. Human ADAM33 refers to a testis cDNA clone that does not contain a complete open reading frame, but portions of the predicted protein are similar to Xenopus laevis ADAM13. RESULTS: In a 48 kb region of mouse DNA adjacent to the Attractin gene on mouse chromosome 2, we identified sequences very similar to human ADAM33. A full-length mouse cDNA was identified by a combination of gene prediction programs and RT-PCR, and the probable full-length human cDNA was identified by comparison to human genomic sequence in the homologous region on chromosome 20p13. Mouse ADAM33 is 44% identical to Xenopus laevis ADAM13, however a phylogenetic alignment and consideration of functional domains suggests that the two genes are not orthologous. Mouse Adam33 is widely expressed, most highly in the adult brain, heart, kidney, lung and testis. CONCLUSIONS: While mouse ADAM33 is similar to Xenopus ADAM13 in sequence, further examination of its embryonic expression pattern, catalytic activity and protein interactions will be required to assess the functional relationship between these two proteins. Adam33 is expressed in the mouse adult brain and could play a role in complex processes that require cell-cell communication.