P. Scott White

TNF-α Promoter Polymorphisms and Susceptibility to Human Papillomavirus 16–Associated Cervical Cancer

BACKGROUND Polymorphisms in the TNF-alpha promoter region have recently been shown to be associated with susceptibility to cervical cancer. Some polymorphisms have been reported to influence transcription for this cytokine. Altered local levels in the cervix may influence an individuals immune response, thereby affecting persistence of human papillomavirus (HPV) 16 infection, a primary etiological factor for cervical cancer. METHODS AND RESULTS The association of 11 TNF-alpha single-nucleotide polymorphisms (SNPs) with susceptibility to HPV16-associated cervical cancer was investigated. Sequencing of the TNF-alpha promoter region confirmed 10 SNPs, and 1 previously unreported SNP (161 bp upstream of the transcriptional start site) was discovered. Microsphere-array flow cytometry-based genotyping was performed on 787 samples from Hispanic and non-Hispanic white women (241 from randomly selected control subjects, 205 from HPV16-positive control subjects, and 341 from HPV16-positive subjects with cervical cancer). The genotype distribution of 3 SNPs (-572, -857, and -863) was significantly different between case subjects and control subjects. Analysis of haplotypes, which were computationally inferred from genotype data, also revealed statistically significant differences in haplotype distribution between case subjects and control subjects. CONCLUSIONS We report new associations between several TNF-alpha SNPs and susceptibility to cervical cancer that support the involvement of the TNF- alpha promoter region in development of cervical cancer.

The Systematics and Evolution of the Crocodilia as Suggested by Restriction Endonuclease Analysis of Mitochondrial and Nuclear Ribosomal DNA

We present restriction fragment analyses of mitochondrial and ribosomal DNAs in an effort to resolve some critical systematic questions among extant members of the order Crocodilia. This paper concentrates on interspecific relationships among all the species of the circumtropical genus Crocodylus (the true crocodiles), the relationship of Crocodylus to Osteolaemus (the dwarf-African crocodile) and tests the hypothesis that the two gharial genera, Gavialis and Tomistoma, are more closely related to one another than to any other group of living crocodilians. This first extensive molecular phylogeny of the true crocodiles not only aligns some species that are found in the same hemisphere but also places organisms that currently have disjunct distributions in the same clade. We also include a method of analysis (compatible parsimony) for restriction fragment data that appears to combine compatibility and parsimony criteria.

Exordium for DNA Codes

We describe how deletion-correcting codes may be enhanced to yield codes with double-strand DNA-sequence codewords. This enhancement involves abstractions of the pertinent aspects of DNA; it nevertheless ensures specificity of binding for all pairs of single strands derived from its codewords—the key desideratum of DNA codes– i.e. with binding feasible only between reverse complementary strands. We defer discussing the combinatorial-optimization superincumbencies of code construction. Generalization of deletion similarity to an optimal sequence-alignment score could readily effect advantageous improvements (Kaderali, Masters Thesis, Informatics, U. Köln, 2001) but would render the combinatorics opaque. We mention motivating applications of DNA codes.

A rapid multiplex assay for nucleic acid-based diagnostics

We have developed a rapid (under 4 hours), multiplex, nucleic acid assay, adapted to a microsphere array detection platform. We call this assay multiplex oligonucleotide ligation-PCR (MOL-PCR). Unlike other ligation-based assays that require multiple steps, our protocol consists of a single tube reaction, followed by hybridization to a Luminex microsphere array for detection. We demonstrate the ability of this assay to simultaneously detect diverse nucleic acid signatures (e.g., unique sequences, single nucleotide polymorphisms) in a single multiplex reaction. Detection probes consist of modular components that enable target detection, probe amplification, and subsequent capture onto microsphere arrays. To demonstrate the utility of our assay, we applied it to the detection of three biothreat agents, B. anthracis, Y. pestis, and F. tularensis. Combined with the ease and robustness of this assay, the results presented here show a strong potential of our assay for use in diagnostics and surveillance.

SNPsFinder---a web-based application for genome-wide discovery of single nucleotide polymorphisms in microbial genomes

UNLABELLED Single nucleotide polymorphisms (SNPs) are the most abundant form of genetic variations in closely related microbial species, strains or isolates. Some SNPs confer selective advantages for microbial pathogens during infection and many others are powerful genetic markers for distinguishing closely related strains or isolates that could not be distinguished otherwise. To facilitate SNP discovery in microbial genomes, we have developed a web-based application, SNPsFinder, for genome-wide identification of SNPs. SNPsFinder takes multiple genome sequences as input to identify SNPs within homologous regions. It can also take contig sequences and sequence quality scores from ongoing sequencing projects for SNP prediction. SNPsFinder will use genome sequence annotation if available and map the predicted SNP regions to known genes or regions to assist further evaluation of the predicted SNPs for their functional significance. SNPsFinder can generate PCR primers for all predicted SNP regions according to users input parameters to facilitate experimental validation. The results from SNPsFinder analysis are accessible through the World Wide Web. AVAILABILITY The SNPsFinder program is available at http://snpsfinder.lanl.gov/. SUPPLEMENTARY INFORMATION The users manual is available at http://snpsfinder.lanl.gov/UsersManual/

Variation in HLA Class I Antigen-Processing Genes and Susceptibility to Human Papillomavirus Type 16—Associated Cervical Cancer

BACKGROUND Persistent infection with human papillomavirus type 16 (HPV16) is a primary etiological factor for the development of cervical cancer. Genes involved in antigen processing influence both the repertoire of antigens presented by HPV16-infected cells and the nature of HPV16-specific immune responses. Genetic variation in these genes may affect protein structure and function and, consequently, the ability of an individual to clear HPV infection. METHODS Thirty-five single-nucleotide polymorphisms (SNPs) in 5 genes (LMP2, TAP1, LMP7, TAP2, and Tapasin) were investigated for association with susceptibility to HPV16-associated cervical cancer. Sequencing of these genes resulted in the discovery of 15 previously unreported SNPs. Microsphere-array flow cytometry-based genotyping was conducted on 787 samples from Hispanic and non-Hispanic white women (241 randomly selected control subjects, 205 HPV16-positive control subjects, and 341 HPV16-positive case subjects with cervical cancer). RESULTS For 9 SNPs, 8 of which had not previously been reported in the context of cervical cancer, there were statistically significant differences between the genotype distribution in case subjects and that in control subjects. Haplotype analysis of 3 haplotype blocks revealed 3 haplotypes with significant differences in frequency in case-control comparisons. Both HPV16-specific and non-type-specific differences in genotype distribution were seen. CONCLUSIONS Genes involved in antigen processing for HLA class I presentation may contribute to susceptibility to cervical cancer.

Development of rationally designed nucleic acid signatures for microbial pathogens.

The detection and identification of microbial pathogens are critical challenges in clinical medicine and public health surveillance. Advances in genome analysis technology are providing an unprecedented amount of information about bacterial and viral organisms, and hold great potential for pathogen detection and identification. In this paper, a rational approach to the development and application of nucleic acid signatures is described based on phylogenetically informative sequence features, especially single nucleotide polymorphisms. The computational tools that are available to enable the development of the next generation of microbial molecular signatures for clinical diagnostics and infectious disease surveillance are reviewed and the impact on public health and national security will be discussed.

Simultaneous pathogen detection and antibiotic resistance characterization using SNP-based multiplexed oligonucleotide ligation-PCR (MOL-PCR).

Extensive use of antibiotics in both public health and animal husbandry has resulted in rapid emergence of antibiotic resistance in almost all human pathogens, including biothreat pathogens. Antibiotic resistance has thus become a major concern for both public health and national security. We developed multiplexed assays for rapid, simultaneous pathogen detection and characterization of ciprofloxacin and doxycycline resistance in Bacillus anthracis, Yersinia pestis, and Francisella tularensis. These assays are SNP-based and use Multiplexed Oligonucleotide Ligation-PCR (MOL-PCR). The MOL-PCR assay chemistry and MOLigo probe design process are presented. A web-based tool - MOLigoDesigner (http://MOLigoDesigner.lanl.gov) was developed to facilitate the probe design. All probes were experimentally validated individually and in multiplexed assays, and minimal sets of multiplexed MOLigo probes were identified for simultaneous pathogen detection and antibiotic resistance characterization.

3rd International Meeting on Single Nucleotide Polymorphism and Complex Genome Analysis: SNPs: 'some notable progress'.

Fervent activities for the collection and exploitation of single nucleotide polymorphism (SNP) data continue, amid concerns about their real utility. The desire to understand complex disease aetiology remains a key driving force for this activity. Recent developments provided a level of cautious optimism not seen in previous International Meetings on Single Nucleotide Polymorphism and Complex Genome Analysis. The 3rd such meeting, held 8–11 September 2000 in Taos, New Mexico, covered research on technologies for SNP scoring, analytical tools for using SNPs to map disease genes, examples from researchers using SNPs for specific disease studies, and databases and tools for facilitating these activities. Studies of human history, and a range of studies upon model organisms were also represented. Whilst the transition from technology oriented work (methods, discovery, etc.) to successful biological application is occurring relatively slowly, a clear trend in this direction is now apparent, and it will surely gain momentum in future months and years. Many fundamental properties of SNPs remain unknown, and many other basic questions are still unanswered, but the field is moving forward on all necessary fronts, promising exciting advances just around the corner.