Complete Biochemical Characterization of Pantaphos Biosynthesis Highlights an Unusual Role for a SAM-Dependent Methyltransferase.

Pantaphos is small molecule virulence factor made by the plant pathogen Pantoea ananatis. An 11 gene operon, designated hvr for high virulence, is required for production of this phosphonic acid natural product, but the metabolic steps used in its production have yet to be established. Herein, we determine the complete biosynthetic pathway using a combination of bioinformatics, in vitro biochemistry and in vivo heterologous expression. Only 6 of the 11 hvr genes are needed to produce pantaphos, while a seventh is likely to be required for export. Surprisingly, the pathway involves a series of O-methylated intermediates, which are then hydrolyzed to produce the final product. The methylated intermediates are produced by an irreversible S-adenosylmethione (SAM)-dependent methyltransferase that is required to drive a thermodynamically unfavorable dehydration in the preceding step, a function not previously attributed to members of this enzyme class. Methylation of pantaphos by the same enzyme is also likely to limit its toxicity in the producing organism. The pathway also involves a novel flavin-dependent monooxygenase that differs from homologous proteins due to its endogenous flavin-reductase activity. Heterologous production of pantaphos by Escherichia coli strains expressing the minimal gene set strongly supports the in vitro biochemical data.

antiSMASH 7.0: new and improved predictions for detection, regulation, chemical structures and visualisation.

Microorganisms produce small bioactive compounds as part of their secondary or specialised metabolism. Often, such metabolites have antimicrobial, anticancer, antifungal, antiviral or other bio-activities and thus play an important role for applications in medicine and agriculture. In the past decade, genome mining has become a widely-used method to explore, access, and analyse the available biodiversity of these compounds. Since 2011, the 'antibiotics and secondary metabolite analysis shell-antiSMASH' (https://antismash.secondarymetabolites.org/) has supported researchers in their microbial genome mining tasks, both as a free to use web server and as a standalone tool under an OSI-approved open source licence. It is currently the most widely used tool for detecting and characterising biosynthetic gene clusters (BGCs) in archaea, bacteria, and fungi. Here, we present the updated version 7 of antiSMASH. antiSMASH 7 increases the number of supported cluster types from 71 to 81, as well as containing improvements in the areas of chemical structure prediction, enzymatic assembly-line visualisation and gene cluster regulation.

A Novel Pathway for Biosynthesis of the Herbicidal Phosphonate Natural Product Phosphonothrixin Is Widespread in Actinobacteria.

Phosphonothrixin is an herbicidal phosphonate natural product with an unusual, branched carbon skeleton. Bioinformatic analyses of the ftx gene cluster, which is responsible for synthesis of the compound, suggest that early steps of the biosynthetic pathway, up to production of the intermediate 2,3-dihydroxypropylphosphonic acid (DHPPA) are identical to those of the unrelated phosphonate natural product valinophos. This conclusion was strongly supported by the observation of biosynthetic intermediates from the shared pathway in spent media from two phosphonothrixin producing strains. Biochemical characterization of ftx-encoded proteins confirmed these early steps, as well as subsequent steps involving the oxidation of DHPPA to 3-hydroxy-2-oxopropylphosphonate and its conversion to phosphonothrixin by the combined action of an unusual heterodimeric, thiamine-pyrophosphate (TPP)-dependent ketotransferase and a TPP-dependent acetolactate synthase. The frequent observation of ftx-like gene clusters within actinobacteria suggests that production of compounds related to phosphonothrixin is common within these bacteria. IMPORTANCE Phosphonic acid natural products, such as phosphonothrixin, have great potential for biomedical and agricultural applications; however, discovery and development of these compounds requires detailed knowledge of the metabolism involved in their biosynthesis. The studies reported here reveal the biochemical pathway phosphonothrixin production, which enhances our ability to design strains that overproduce this potentially useful herbicide. This knowledge also improves our ability to predict the products of related biosynthetic gene clusters and the functions of homologous enzymes.

A Novel Biosynthetic Gene Cluster Across the Pantoea Species Complex Is Important for Pathogenicity in Onion.

Onion center rot is caused by at least four species of genus Pantoea (P. ananatis, P. agglomerans, P. allii, and P. stewartii subsp. indologenes). Critical onion pathogenicity determinants for P. ananatis were recently described, but whether those determinants are common among other onion-pathogenic Pantoea species remains unknown. In this work, we report onion pathogenicity determinants in P. stewartii subsp. indologenes and P. allii. We identified two distinct secondary metabolite biosynthetic gene clusters present separately in different strains of onion-pathogenic P. stewartii subsp. indologenes. One cluster is similar to the previously described HiVir phosphonate biosynthetic cluster identified in P. ananatis and another is a novel putative phosphonate biosynthetic gene cluster, which we named Halophos. The Halophos gene cluster was also identified in P. allii strains. Both clusters are predicted to be phosphonate biosynthetic clusters based on the presence of a characteristic phosphoenolpyruvate phosphomutase (pepM) gene. The deletion of the pepM gene from either HiVir or Halophos clusters in P. stewartii subsp. indologenes caused loss of necrosis on onion leaves and red onion scales and resulted in significantly lower bacterial populations compared with the corresponding wild-type and complemented strains. Seven (halB to halH) of 11 genes (halA to halK) in the Halophos gene cluster are required for onion necrosis phenotypes. The onion nonpathogenic strain PNA15-2 (P. stewartii subsp. indologenes) gained the capacity to cause foliar necrosis on onion via exogenous expression of a minimal seven-gene Halophos cluster (genes halB to halH). Furthermore, cell-free culture filtrates of PNA14-12 expressing the intact Halophos gene cluster caused necrosis on onion leaves consistent with the presence of a secreted toxin. Based on the similarity of proteins to those with experimentally determined functions, we are able to predict most of the steps in Halophos biosynthesis. Together, these observations indicate that production of the toxin phosphonate seems sufficient to account for virulence of a variety of different Pantoea strains, although strains differ in possessing a single but distinct phosphonate biosynthetic cluster. Overall, this is the first report of onion pathogenicity determinants in P. stewartii subsp. indologenes and P. allii. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Genetic Methods and Construction of Chromosomal Mutations in Methanogenic Archaea.

Genetic manipulation through markerless exchange enables the modification of several genomic regions without leaving a selection marker in the genome. Here, a method using hpt coding for hypoxanthine phosphoribosyltransferase as a counter selectable marker is described. For Methanosarcina species a chromosomal deletion of the hpt gene is firstly generated, which confers resistance to the purine analogue 8-aza-2,6-diaminopurine (8-ADP). In a second step, the reintroduction of the hpt gene on a plasmid leads to a selectable loss of 8-ADP resistance after a homologous recombination event (pop-in). A subsequent pop-out event restores the 8-ADP resistance and can generate chromosomal mutants with frequencies of about 50%.

Discovery, structure and mechanism of a tetraether lipid synthase.

Archaea synthesize isoprenoid-based ether-linked membrane lipids, which enable them to withstand extreme environmental conditions, such as high temperatures, high salinity, and low or high pH values1-5. In some archaea, such as Methanocaldococcus jannaschii, these lipids are further modified by forming carbon-carbon bonds between the termini of two lipid tails within one glycerophospholipid to generate the macrocyclic archaeol or forming two carbon-carbon bonds between the termini of two lipid tails from two glycerophospholipids to generate the macrocycle glycerol dibiphytanyl glycerol tetraether (GDGT)1,2. GDGT contains two 40-carbon lipid chains (biphytanyl chains) that span both leaflets of the membrane, providing enhanced stability to extreme conditions. How these specialized lipids are formed has puzzled scientists for decades. The reaction necessitates the coupling of two completely inert sp3-hybridized carbon centres, which, to our knowledge, has not been observed in nature. Here we show that the gene product of mj0619 from M. jannaschii, which encodes a radical S-adenosylmethionine enzyme, is responsible for biphytanyl chain formation during synthesis of both the macrocyclic archaeol and GDGT membrane lipids6. Structures of the enzyme show the presence of four metallocofactors: three [Fe4S4] clusters and one mononuclear rubredoxin-like iron ion. In vitro mechanistic studies show that Csp3-Csp3 bond formation takes place on fully saturated archaeal lipid substrates and involves an intermediate bond between the substrate carbon and a sulfur of one of the [Fe4S4] clusters. Our results not only establish the biosynthetic route for tetraether formation but also improve the use of GDGT in GDGT-based paleoclimatology indices7-10.

Incomplete Penetrance of Population-Based Genetic Screening Results in Electronic Health Record.

The clinical value of population-based genetic screening projects depends on the actions taken on the findings. The Healthy Nevada Project (HNP) is an all-comer genetic screening and research project based in northern Nevada. HNP participants with CDC Tier 1 findings of hereditary breast and ovarian cancer syndrome (HBOC), Lynch syndrome (LS), or familial hypercholesterolemia (FH) are notified and provided with genetic counseling. However, the HNP subsequently takes a "hands-off" approach: it is the responsibility of notified participants to share their findings with their healthcare providers, and providers are expected to implement the recommended action plans. Thus, the HNP presents an opportunity to evaluate the efficiency of participant and provider responses to notification of important genetic findings, using electronic health records (EHRs) at Renown Health (a large regional hospital in northern Nevada). Out of 520 HNP participants with findings, we identified 250 participants who were notified of their findings and who had an EHR. 107 of these participants responded to a survey, with 76 (71%) indicating that they had shared their findings with their healthcare providers. However, a sufficiently specific genetic diagnosis appeared in the EHRs and problem lists of only 22 and 10%, respectively, of participants without prior knowledge. Furthermore, review of participant EHRs provided evidence of possible relevant changes in clinical care for only a handful of participants. Up to 19% of participants would have benefited from earlier screening due to prior presentation of their condition. These results suggest that continuous support for both participants and their providers is necessary to maximize the benefit of population-based genetic screening. We recommend that genetic screening projects require participants' consent to directly document their genetic findings in their EHRs. Additionally, we recommend that they provide healthcare providers with ongoing training regarding documentation of findings and with clinical decision support regarding subsequent care.

Investigating Abiotic and Biotic Mechanisms of Pyrite Reduction.

Pyrite (FeS2) has a very low solubility and therefore has historically been considered a sink for iron (Fe) and sulfur (S) and unavailable to biology in the absence of oxygen and oxidative weathering. Anaerobic methanogens were recently shown to reduce FeS2 and assimilate Fe and S reduction products to meet nutrient demands. However, the mechanism of FeS2 mineral reduction and the forms of Fe and S assimilated by methanogens remained unclear. Thermodynamic calculations described herein indicate that H2 at aqueous concentrations as low as 10-10 M favors the reduction of FeS2, with sulfide (HS-) and pyrrhotite (Fe1- x S) as products; abiotic laboratory experiments confirmed the reduction of FeS2 with dissolved H2 concentrations greater than 1.98 × 10-4 M H2. Growth studies of Methanosarcina barkeri provided with FeS2 as the sole source of Fe and S resulted in H2 production but at concentrations too low to drive abiotic FeS2 reduction, based on abiotic laboratory experimental data. A strain of M. barkeri with deletions in all [NiFe]-hydrogenases maintained the ability to reduce FeS2 during growth, providing further evidence that extracellular electron transport (EET) to FeS2 does not involve H2 or [NiFe]-hydrogenases. Physical contact between cells and FeS2 was required for mineral reduction but was not required to obtain Fe and S from dissolution products. The addition of a synthetic electron shuttle, anthraquinone-2,6-disulfonate, allowed for biological reduction of FeS2 when physical contact between cells and FeS2 was prohibited, indicating that exogenous electron shuttles can mediate FeS2 reduction. Transcriptomics experiments revealed upregulation of several cytoplasmic oxidoreductases during growth of M. barkeri on FeS2, which may indicate involvement in provisioning low potential electrons for EET to FeS2. Collectively, the data presented herein indicate that reduction of insoluble FeS2 by M. barkeri occurred via electron transfer from the cell surface to the mineral surface resulting in the generation of soluble HS- and mineral-associated Fe1- x S. Solubilized Fe(II), but not HS-, from mineral-associated Fe1- x S reacts with aqueous HS- yielding aqueous iron sulfur clusters (FeS aq ) that likely serve as the Fe and S source for methanogen growth and activity. FeS aq nucleation and subsequent precipitation on the surface of cells may result in accelerated EET to FeS2, resulting in positive feedback between cell activity and FeS2 reduction.

An Unusual Oxidative Rearrangement Catalyzed by a Divergent Member of the 2-Oxoglutarate-Dependent Dioxygenase Superfamily during Biosynthesis of Dehydrofosmidomycin.

The biosynthesis of the natural product dehydrofosmidomycin involves an unusual transformation in which 2-(trimethylamino)ethylphosphonate is rearranged, desaturated and demethylated by the enzyme DfmD, a divergent member of the 2-oxoglutarate-dependent dioxygenase superfamily. Although other members of this enzyme family catalyze superficially similar transformations, the combination of all three reactions in a single enzyme has not previously been observed. By characterizing the products of in vitro reactions with labeled and unlabeled substrates, we show that DfmD performs this transformation in two steps, with the first involving desaturation of the substrate to form 2-(trimethylamino)vinylphosphonate, and the second involving rearrangement and demethylation to form methyldehydrofosmidomycin. These data reveal significant differences from the desaturation and rearrangement reactions catalyzed by other family members.

Role of internal loop dynamics in antibiotic permeability of outer membrane porins.

Gram-negative bacteria pose a serious public health concern due to resistance to many antibiotics, caused by the low permeability of their outer membrane (OM). Effective antibiotics use porins in the OM to reach the interior of the cell; thus, understanding permeation properties of OM porins is instrumental to rationally develop broad-spectrum antibiotics. A functionally important feature of OM porins is undergoing open-closed transitions that modulate their transport properties. To characterize the molecular basis of these transitions, we performed an extensive set of molecular dynamics (MD) simulations of Escherichia coli OM porin OmpF. Markov-state analysis revealed that large-scale motion of an internal loop, L3, underlies the transition between energetically stable open and closed states. The conformation of L3 is controlled by H bonds between highly conserved acidic residues on the loop and basic residues on the OmpF ß-barrel. Mutation of key residues important for the loop's conformation shifts the equilibrium between open and closed states and regulates translocation of permeants (ions and antibiotics), as observed in the simulations and validated by our whole-cell accumulation assay. Notably, one mutant system G119D, which we find to favor the closed state, has been reported in clinically resistant bacterial strains. Overall, our accumulated ∼200 µs of simulation data (the wild type and mutants) along with experimental assays suggest the involvement of internal loop dynamics in permeability of OM porins and antibiotic resistance in Gram-negative bacteria.

Rationalizing the generation of broad spectrum antibiotics with the addition of a positive charge.

Antibiotic resistance of Gram-negative bacteria is largely attributed to the low permeability of their outer membrane (OM). Recently, we disclosed the eNTRy rules, a key lesson of which is that the introduction of a primary amine enhances OM permeation in certain contexts. To understand the molecular basis for this finding, we perform an extensive set of molecular dynamics (MD) simulations and free energy calculations comparing the permeation of aminated and amine-free antibiotic derivatives through the most abundant OM porin of E. coli, OmpF. To improve sampling of conformationally flexible drugs in MD simulations, we developed a novel, Monte Carlo and graph theory based algorithm to probe more efficiently the rotational and translational degrees of freedom visited during the permeation of the antibiotic molecule through OmpF. The resulting pathways were then used for free-energy calculations, revealing a lower barrier against the permeation of the aminated compound, substantiating its greater OM permeability. Further analysis revealed that the amine facilitates permeation by enabling the antibiotic to align its dipole to the luminal electric field of the porin and form favorable electrostatic interactions with specific, highly-conserved charged residues. The importance of these interactions in permeation was further validated with experimental mutagenesis and whole cell accumulation assays. Overall, this study provides insights on the importance of the primary amine for antibiotic permeation into Gram-negative pathogens that could help the design of future antibiotics. We also offer a new computational approach for calculating free-energy of processes where relevant molecular conformations cannot be efficiently captured.

SARS-CoV-2 test positivity rate in Reno, Nevada: association with PM2.5 during the 2020 wildfire smoke events in the western United States.

BACKGROUND: Air pollution has been linked to increased susceptibility to SARS-CoV-2. Thus, it has been suggested that wildfire smoke events may exacerbate the COVID-19 pandemic. OBJECTIVES: Our goal was to examine whether wildfire smoke from the 2020 wildfires in the western United States was associated with an increased rate of SARS-CoV-2 infections in Reno, Nevada. METHODS: We conducted a time-series analysis using generalized additive models to examine the relationship between the SARS-CoV-2 test positivity rate at a large regional hospital in Reno and ambient PM2.5 from 15 May to 20 Oct 2020. RESULTS: We found that a 10 µg/m3 increase in the 7-day average PM2.5 concentration was associated with a 6.3% relative increase in the SARS-CoV-2 test positivity rate, with a 95% confidence interval (CI) of 2.5 to 10.3%. This corresponded to an estimated 17.7% (CI: 14.4-20.1%) increase in the number of cases during the time period most affected by wildfire smoke, from 16 Aug to 10 Oct. SIGNIFICANCE: Wildfire smoke may have greatly increased the number of COVID-19 cases in Reno. Thus, our results substantiate the role of air pollution in exacerbating the pandemic and can help guide the development of public preparedness policies in areas affected by wildfire smoke, as wildfires are likely to coincide with the COVID-19 pandemic in 2021.

A Phosphonate Natural Product Made by Pantoea ananatis is Necessary and Sufficient for the Hallmark Lesions of Onion Center Rot.

Pantoea ananatis is the primary cause of onion center rot. Genetic data suggest that a phosphonic acid natural product is required for pathogenesis; however, the nature of the molecule is unknown. Here, we show that P. ananatis produces at least three phosphonates, two of which were purified and structurally characterized. The first, designated pantaphos, was shown to be 2-(hydroxy[phosphono]methyl)maleate; the second, a probable biosynthetic precursor, was shown to be 2-(phosphonomethyl)maleate. Purified pantaphos is both necessary and sufficient for the hallmark lesions of onion center rot. Moreover, when tested against mustard seedlings, the phytotoxic activity of pantaphos was comparable to the widely used herbicides glyphosate and phosphinothricin. Pantaphos was also active against a variety of human cell lines but was significantly more toxic to glioblastoma cells. Pantaphos showed little activity when tested against a variety of bacteria and fungi.IMPORTANCEPantoea ananatis is a significant plant pathogen that targets a number of important crops, a problem that is compounded by the absence of effective treatments to prevent its spread. Our identification of pantaphos as the key virulence factor in onion center rot suggests a variety of approaches that could be employed to address this significant plant disease. Moreover, the general phytotoxicity of the molecule suggests that it could be developed into an effective herbicide to counter the alarming rise in herbicide-resistant weeds.

Genome Mining and Metabolomics Uncover a Rare d-Capreomycidine Containing Natural Product and Its Biosynthetic Gene Cluster.

We report the metabolomics-driven genome mining of a new cyclic-guanidino incorporating non-ribosomal peptide synthetase (NRPS) gene cluster and full structure elucidation of its associated hexapeptide product, faulknamycin. Structural studies unveiled that this natural product contained the previously unknown (R,S)-stereoisomer of capreomycidine, d-capreomycidine. Furthermore, heterologous expression of the identified gene cluster successfully reproduces faulknamycin production without an observed homologue of VioD, the pyridoxal phosphate (PLP)-dependent enzyme found in all previous l-capreomycidine biosynthesis. An alternative NRPS-dependent pathway for d-capreomycidine biosynthesis is proposed.

Particulate matter and emergency visits for asthma: a time-series study of their association in the presence and absence of wildfire smoke in Reno, Nevada, 2013-2018.

BACKGROUND: Health risks due to particulate matter (PM) from wildfires may differ from risk due to PM from other sources. In places frequently subjected to wildfire smoke, such as Reno, Nevada, it is critical to determine whether wildfire PM poses unique risks. Our goal was to quantify the difference in the association of adverse asthma events with PM on days when wildfire smoke was present versus days when wildfire smoke was not present. METHODS: We obtained counts of visits for asthma at emergency departments and urgent care centers from a large regional healthcare system in Reno for the years 2013-2018. We also obtained dates when wildfire smoke was present from the Washoe County Health District Air Quality Management Division. We then examined whether the presence of wildfire smoke modified the association of PM2.5, PM10-2.5, and PM10 with asthma visits using generalized additive models. We improved on previous studies by excluding wildfire-smoke days where the PM concentration exceeded the maximum PM concentration on other days, thus accounting for possible nonlinearity in the association between PM concentration and asthma visits. RESULTS: Air quality was affected by wildfire smoke on 188 days between 2013 and 2018. We found that the presence of wildfire smoke increased the association of a 5 µg/m3 increase in daily and three-day averages of PM2.5 with asthma visits by 6.1% (95% confidence interval (CI): 2.1-10.3%) and 6.8% (CI: 1.2-12.7%), respectively. Similarly, the presence of wildfire smoke increased the association of a 5 µg/m3 increase in daily and three-day averages of PM10 with asthma visits by 5.5% (CI: 2.5-8.6%) and 7.2% (CI: 2.6-12.0%), respectively. We did not observe any significant increases in association for PM10-2.5 or for seven-day averages of PM2.5 and PM10. CONCLUSIONS: Since we found significantly stronger associations of PM2.5 and PM10 with asthma visits when wildfire smoke was present, our results suggest that wildfire PM is more hazardous than non-wildfire PM for patients with asthma.

Functional interactions between posttranslationally modified amino acids of methyl-coenzyme M reductase in Methanosarcina acetivorans.

The enzyme methyl-coenzyme M reductase (MCR) plays an important role in mediating global levels of methane by catalyzing a reversible reaction that leads to the production or consumption of this potent greenhouse gas in methanogenic and methanotrophic archaea. In methanogenic archaea, the alpha subunit of MCR (McrA) typically contains four to six posttranslationally modified amino acids near the active site. Recent studies have identified enzymes performing two of these modifications (thioglycine and 5-[S]-methylarginine), yet little is known about the formation and function of the remaining posttranslationally modified residues. Here, we provide in vivo evidence that a dedicated S-adenosylmethionine-dependent methyltransferase encoded by a gene we designated methylcysteine modification (mcmA) is responsible for formation of S-methylcysteine in Methanosarcina acetivorans McrA. Phenotypic analysis of mutants incapable of cysteine methylation suggests that the S-methylcysteine residue might play a role in adaption to mesophilic conditions. To examine the interactions between the S-methylcysteine residue and the previously characterized thioglycine, 5-(S)-methylarginine modifications, we generated M. acetivorans mutants lacking the three known modification genes in all possible combinations. Phenotypic analyses revealed complex, physiologically relevant interactions between the modified residues, which alter the thermal stability of MCR in a combinatorial fashion that is not readily predictable from the phenotypes of single mutants. High-resolution crystal structures of inactive MCR lacking the modified amino acids were indistinguishable from the fully modified enzyme, suggesting that interactions between the posttranslationally modified residues do not exert a major influence on the static structure of the enzyme but rather serve to fine-tune the activity and efficiency of MCR.

Acute associations between PM2.5 and ozone concentrations and asthma exacerbations among patients with and without allergic comorbidities.

Acute effects of outdoor air pollution on asthma exacerbations may vary by asthma phenotype (allergic vs nonallergic). Associations of ambient PM2.5 and ozone concentrations with acute asthma visits (office, urgent, emergency, and hospitalization) were investigated using electronic medical records. International Classification of Disease codes were used to identify asthmatics, and classify them based on the presence or absence of an allergic comorbidity in their medical records. Daily 24-h average PM2.5, 8-h maximum ozone, and mean temperature were obtained from a centralized monitor. Using a time-stratified case-crossover approach, pollutant concentrations were modeled using moving averages and distributed lag nonlinear models (lag 0-6) to examine lag associations and nonlinear concentration-response. The adjusted odds ratios for a 10 µg/m3 increase in 3-day moving average (lag 0-2) PM2.5 in the two-pollutant models among patients with and without allergic comorbidities were 1.10 (95% confidence interval [CI]: 1.07, 1.13) and 1.05 (95% CI: 1.02, 1.09), respectively; and for a 20 ppb increase in 3-day moving average (lag 0-2) ozone were 1.08 (95% CI: 1.02, 1.14) and 1.00 (95% CI: 0.95, 1.05), respectively. Estimated odds ratios among patients with allergic comorbidities were consistently higher across age, sex, and temperature categories. Asthmatics with an allergic comorbidity may be more susceptible to ambient PM2.5 and ozone.

Genome-wide rare variant analysis for thousands of phenotypes in over 70,000 exomes from two cohorts.

Understanding the impact of rare variants is essential to understanding human health. We analyze rare (MAF < 0.1%) variants against 4264 phenotypes in 49,960 exome-sequenced individuals from the UK Biobank and 1934 phenotypes (1821 overlapping with UK Biobank) in 21,866 members of the Healthy Nevada Project (HNP) cohort who underwent Exome + sequencing at Helix. After using our rare-variant-tailored methodology to reduce test statistic inflation, we identify 64 statistically significant gene-based associations in our meta-analysis of the two cohorts and 37 for phenotypes available in only one cohort. Singletons make significant contributions to our results, and the vast majority of the associations could not have been identified with a genotyping chip. Our results are available for interactive browsing in a webapp (https://ukb.research.helix.com). This comprehensive analysis illustrates the biological value of large, deeply phenotyped cohorts of unselected populations coupled with NGS data.

A Comprehensive Genome-Wide and Phenome-Wide Examination of BMI and Obesity in a Northern Nevadan Cohort.

The aggregation of Electronic Health Records (EHR) and personalized genetics leads to powerful discoveries relevant to population health. Here we perform genome-wide association studies (GWAS) and accompanying phenome-wide association studies (PheWAS) to validate phenotype-genotype associations of BMI, and to a greater extent, severe Class 2 obesity, using comprehensive diagnostic and clinical data from the EHR database of our cohort. Three GWASs of 500,000 variants on the Illumina platform of 6,645 Healthy Nevada participants identified several published and novel variants that affect BMI and obesity. Each GWAS was followed with two independent PheWASs to examine associations between extensive phenotypes (incidence of diagnoses, condition, or disease), significant SNPs, BMI, and incidence of extreme obesity. The first GWAS examines associations with BMI in a cohort with no type 2 diabetics, focusing exclusively on BMI. The second GWAS examines associations with BMI in a cohort that includes type 2 diabetics. In the second GWAS, type 2 diabetes is a comorbidity, and thus becomes a covariate in the statistical model. The intersection of significant variants of these two studies is surprising. The third GWAS is a case vs. control study, with cases defined as extremely obese (Class 2 or 3 obesity), and controls defined as participants with BMI between 18.5 and 25. This last GWAS identifies strong associations with extreme obesity, including established variants in the FTO and NEGR1 genes, as well as loci not yet linked to obesity. The PheWASs validate published associations between BMI and extreme obesity and incidence of specific diagnoses and conditions, yet also highlight novel links. This study emphasizes the importance of our extensive longitudinal EHR database to validate known associations and identify putative novel links with BMI and obesity.