Christine Fong

PGAT: a multistrain analysis resource for microbial genomes

Motivation: The Prokaryotic-genome Analysis Tool (PGAT) is a web-based database application for comparing gene content and sequence across multiple microbial genomes facilitating the discovery of genetic differences that may explain observed phenotypes. PGAT supports database queries to identify genes that are present or absent in user-selected genomes, comparison of sequence polymorphisms in sets of orthologous genes, multigenome display of regions surrounding a query gene, comparison of the distribution of genes in metabolic pathways and manual community annotation. Availability and Implementation:The PGAT website may be accessed at http://nwrce.org/pgat. Contact: mbrittna@uw.edu

Comparison of Francisella tularensis genomes reveals evolutionary events associated with the emergence of human pathogenic strains

BackgroundFrancisella tularensis subspecies tularensis and holarctica are pathogenic to humans, whereas the two other subspecies, novicida and mediasiatica, rarely cause disease. To uncover the factors that allow subspecies tularensis and holarctica to be pathogenic to humans, we compared their genome sequences with the genome sequence of Francisella tularensis subspecies novicida U112, which is nonpathogenic to humans.ResultsComparison of the genomes of human pathogenic Francisella strains with the genome of U112 identifies genes specific to the human pathogenic strains and reveals pseudogenes that previously were unidentified. In addition, this analysis provides a coarse chronology of the evolutionary events that took place during the emergence of the human pathogenic strains. Genomic rearrangements at the level of insertion sequences (IS elements), point mutations, and small indels took place in the human pathogenic strains during and after differentiation from the nonpathogenic strain, resulting in gene inactivation.ConclusionThe chronology of events suggests a substantial role for genetic drift in the formation of pseudogenes in Francisella genomes. Mutations that occurred early in the evolution, however, might have been fixed in the population either because of evolutionary bottlenecks or because they were pathoadaptive (beneficial in the context of infection). Because the structure of Francisella genomes is similar to that of the genomes of other emerging or highly pathogenic bacteria, this evolutionary scenario may be shared by pathogens from other species.

Evolution of Burkholderia pseudomallei in Recurrent Melioidosis

Burkholderia pseudomallei, the etiologic agent of human melioidosis, is capable of causing severe acute infection with overwhelming septicemia leading to death. A high rate of recurrent disease occurs in adult patients, most often due to recrudescence of the initial infecting strain. Pathogen persistence and evolution during such relapsing infections are not well understood. Bacterial cells present in the primary inoculum and in late infections may differ greatly, as has been observed in chronic disease, or they may be genetically similar. To test these alternative models, we conducted whole-genome comparisons of clonal primary and relapse B. pseudomallei isolates recovered six months to six years apart from four adult Thai patients. We found differences within each of the four pairs, and some, including a 330 Kb deletion, affected substantial portions of the genome. Many of the changes were associated with increased antibiotic resistance. We also found evidence of positive selection for deleterious mutations in a TetR family transcriptional regulator from a set of 107 additional B. pseudomallei strains. As part of the study, we sequenced to base-pair accuracy the genome of B. pseudomallei strain 1026b, the model used for genetic studies of B. pseudomallei pathogenesis and antibiotic resistance. Our findings provide new insights into pathogen evolution during long-term infections and have important implications for the development of intervention strategies to combat recurrent melioidosis.

A Genome-wide In Vitro Bacterial-Infection Screen Reveals Human Variation in the Host Response Associated with Inflammatory Disease

Recent progress in cataloguing common genetic variation has made possible genome-wide studies that are beginning to elucidate the causes and consequences of our genetic differences. Approaches that provide a mechanistic understanding of how genetic variants function to alter disease susceptibility and why they were substrates of natural selection would complement other approaches to human-genome analysis. Here we use a novel cell-based screen of bacterial infection to identify human variation in Salmonella-induced cell death. A loss-of-function allele of CARD8, a reported inhibitor of the proinflammatory protease caspase-1, was associated with increased cell death in vitro (p = 0.013). The validity of this association was demonstrated through overexpression of alternative alleles and RNA interference in cells of varying genotype. Comparison of mammalian CARD8 orthologs and examination of variation among different human populations suggest that the increase in infectious-disease burden associated with larger animal groups (i.e., herds and colonies), and possibly human population expansion, may have naturally selected for loss of CARD8. We also find that the loss-of-function CARD8 allele shows a modest association with an increased risk of systemic inflammatory response syndrome in a small study (p = 0.05). Therefore, a by-product of the selected benefit of loss of CARD8 could be increased inflammatory diseases. These results demonstrate the utility of genome-wide cell-based association screens with microbes in the identification of naturally selected variants that can impact human health.

Functional genetic screen of human diversity reveals that a methionine salvage enzyme regulates inflammatory cell death

Genome-wide association studies can identify common differences that contribute to human phenotypic diversity and disease. When genome-wide association studies are combined with approaches that test how variants alter physiology, biological insights can emerge. Here, we used such an approach to reveal regulation of cell death by the methionine salvage pathway. A common SNP associated with reduced expression of a putative methionine salvage pathway dehydratase, apoptotic protease activating factor 1 (APAF1)-interacting protein (APIP), was associated with increased caspase-1–mediated cell death in response to Salmonella. The role of APIP in methionine salvage was confirmed by growth assays with methionine-deficient media and quantitation of the methionine salvage substrate, 5′-methylthioadenosine. Reducing expression of APIP or exogenous addition of 5′-methylthioadenosine increased Salmonellae-induced cell death. Consistent with APIP originally being identified as an inhibitor of caspase-9–dependent apoptosis, the same allele was also associated with increased sensitivity to the chemotherapeutic agent carboplatin. Our results show that common human variation affecting expression of a single gene can alter susceptibility to two distinct cell death programs. Furthermore, the same allele that promotes cell death is associated with improved survival of individuals with systemic inflammatory response syndrome, suggesting a possible evolutionary pressure that may explain the geographic pattern observed for the frequency of this SNP. Our study shows that in vitro association screens of disease-related traits can not only reveal human genetic differences that contribute to disease but also provide unexpected insights into cell biology.

PSAT: A web tool to compare genomic neighborhoods of multiple prokaryotic genomes

BackgroundThe conservation of gene order among prokaryotic genomes can provide valuable insight into gene function, protein interactions, or events by which genomes have evolved. Although some tools are available for visualizing and comparing the order of genes between genomes of study, few support an efficient and organized analysis between large numbers of genomes. The Prokaryotic Sequence homology Analysis Tool (PSAT) is a web tool for comparing gene neighborhoods among multiple prokaryotic genomes.ResultsPSAT utilizes a database that is preloaded with gene annotation, BLAST hit results, and gene-clustering scores designed to help identify regions of conserved gene order. Researchers use the PSAT web interface to find a gene of interest in a reference genome and efficiently retrieve the sequence homologs found in other bacterial genomes. The tool generates a graphic of the genomic neighborhood surrounding the selected gene and the corresponding regions for its homologs in each comparison genome. Homologs in each region are color coded to assist users with analyzing gene order among various genomes. In contrast to common comparative analysis methods that filter sequence homolog data based on alignment score cutoffs, PSAT leverages gene context information for homologs, including those with weak alignment scores, enabling a more sensitive analysis. Features for constraining or ordering results are designed to help researchers browse results from large numbers of comparison genomes in an organized manner. PSAT has been demonstrated to be useful for helping to identify gene orthologs and potential functional gene clusters, and detecting genome modifications that may result in loss of function.ConclusionPSAT allows researchers to investigate the order of genes within local genomic neighborhoods of multiple genomes. A PSAT web server for public use is available for performing analyses on a growing set of reference genomes through any web browser with no client side software setup or installation required. Source code is freely available to researchers interested in setting up a local version of PSAT for analysis of genomes not available through the public server. Access to the public web server and instructions for obtaining source code can be found at http://www.nwrce.org/psat.

GWAS Analyzer

Motivation: Genome-wide association studies are beginning to elucidate how our genetic differences contribute to susceptibility and severity of disease. While computational tools have previously been developed to support various aspects of genome-wide association studies, there is currently a need for informatics solutions that facilitate the integration of data from multiple sources. Results: Here we present GWAS Analyzer, a database driven web-based tool that integrates genotype and phenotype data, association analysis results and genomic annotations from multiple public resources. GWAS Analyzer contains features for browsing these interrelated data, exploring phenotypic values by family or genotype, and filtering association results based on multiple criteria. The utility of the tool has been demonstrated by a genome-wide association study of human in vitro susceptibility to bacterial infection. GWAS Analyzer facilitated management of large sets of phenotype and genotype data, analysis of phenotypic variation and heritability, and most importantly, generation of a refined set of candidate single nucleotide polymorphisms (SNPs). The tool revealed a SNP that was experimentally validated to be associated with increased cell death among Salmonella infected HapMap cell lines. Availability: http://www.nwrce.org/gwas-analyzer Contact: mbrittna@u.washington.edu Supplementary Information: Supplementary data are available at Bioinformatics online.

Evaluating Risk of ESRD in the Urban Poor

The capacity of risk prediction to guide management of CKD in underserved health settings is unknown. We conducted a retrospective cohort study of 28,779 adults with nondialysis-requiring CKD who received health care in two large safety net health systems during 1996-2009 and were followed for ESRD through September of 2011. We developed and evaluated the performance of ESRD risk prediction models using recently proposed criteria designed to inform population health approaches to disease management: proportion of cases followed and proportion that needs to be followed. Overall, 1730 persons progressed to ESRD during follow-up (median follow-up=6.6 years). ESRD risk for time frames up to 5 years was highly concentrated among relatively few individuals. A predictive model using five common variables (age, sex, race, eGFR, and dipstick proteinuria) performed similarly to more complex models incorporating extensive sociodemographic and clinical data. Using this model, 80% of individuals who eventually developed ESRD were among the 5% of cohort members at the highest estimated risk for ESRD at 1 year. Similarly, a program that followed 8% and 13% of individuals at the highest ESRD risk would have included 80% of those who eventually progressed to ESRD at 3 and 5 years, respectively. In this underserved health setting, a simple five-variable model accurately predicts most cases of ESRD that develop within 5 years. Applying risk prediction using a population health approach may improve CKD surveillance and management of vulnerable groups by directing resources to a small subpopulation at highest risk for progressing to ESRD.

Abstract PR05: Using medical informatics to evaluate the risk of colorectal cancer in patients with clinically diagnosed sessile serrated polyps

Background : Recent research suggests that in addition to advanced adenomas, sessile serrated polyps (SSPs) may be important precursors for proximal colon cancer. In this study, we conducted the first large cohort study to evaluate the risk of colorectal cancer (CRC) in patients diagnosed with SSPs through usual care. Methods : The University of Washington Medical Center (UWMC) uses a comprehensive electronic medical records (EMR) system to track patient demographics and health-related information, including diagnoses and procedures for all patients. We used procedure codes to identify a cohort of patients receiving colonoscopies at UWMC from 2003-2013. Natural language processing of text in the final diagnosis section of the pathology report was used to characterize the type of polyps present at each colonoscopy procedure, including non-advanced conventional adenomas, advanced conventional adenomas (defined as having villous histology, high-grade dysplasia, or a diameter ≥ 10 mm), and sessile serrated polyps. These colonoscopy records were then linked to the Puget Sound Surveillance, Epidemiology, and End Results Cancer Registry (SEER) and subsequent EMR data to identify incident CRCs occurring through December 31, 2014 within this cohort. Those who lived outside of the SEER catchment area or who had prior colectomy, inflammatory bowel disease, or CRC were excluded from analyses. Cox proportional hazards regression models were used to calculate adjusted hazard ratios (HRs) and 95% confidence intervals (CIs) comparing the risk of CRC in each polyp group to those who were polyp-free at an index colonoscopy. HR estimates were adjusted for age, sex, race/ethnicity, smoking status, and body mass index. Results : From 2003 through 2013, 32,136 colonoscopies were performed at UWMC, and 17,424 colonoscopies from 14,846 patients met the study inclusion criteria. Of these patients, 8,908 were polyp-free, 4,145 had only non-advanced conventional adenomas, 927 had advanced conventional adenomas and no SSPs, 314 had ≥1 SSP and ≥1 conventional adenoma, and 552 had SSPs and no conventional adenomas at an index colonoscopy. Median follow-up time in the study cohort was 5.8 years, and 66 incident colorectal cancers occurred during the follow-up period. The risk of incident CRC in those with advanced conventional adenomas at their index colonoscopy was significantly higher than those who were polyp-free (HR=3.9; CI: 1.9-7.7). However, there was not a statistically significant difference in the risk of incident CRC between those who were polyp-free at their index colonoscopy and those who had only non-advanced conventional adenomas (HR=1.5; CI: 0.8-2.6), SSPs with conventional adenomas (HR=2.0; CI: 0.5-8.7), or SSPs without conventional adenomas (HR=1.3; CI: 0.3-5.5). Discussion : Despite recent evidence from cross-sectional studies suggesting that SSPs are high-risk precursors for a subset of colorectal cancers, our results indicate that the risk of CRC in patients with clinically-diagnosed SSPs is similar to the risk of CRC in those with non-advanced adenomas. Additional longitudinal studies of SSPs diagnosed through usual care are needed to inform guidelines for the surveillance of patients with SSPs. This abstract is also being presented as Poster B01. Citation Format: Andrea Burnett-Hartman, Polly A. Newcomb, Chan X. Zeng, Yingye Zheng, John M. Inadomi, Christine Fong, Melissa P. Upton, William M. Grady. Using medical informatics to evaluate the risk of colorectal cancer in patients with clinically diagnosed sessile serrated polyps. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr PR05.