Coupled Single-Cell CRISPR Screening and Epigenomic Profiling Reveals Causal Gene Regulatory Networks.

Here, we present Perturb-ATAC, a method that combines multiplexed CRISPR interference or knockout with genome-wide chromatin accessibility profiling in single cells based on the simultaneous detection of CRISPR guide RNAs and open chromatin sites by assay of transposase-accessible chromatin with sequencing (ATAC-seq). We applied Perturb-ATAC to transcription factors (TFs), chromatin-modifying factors, and noncoding RNAs (ncRNAs) in ∼4,300 single cells, encompassing more than 63 genotype-phenotype relationships. Perturb-ATAC in human B lymphocytes uncovered regulators of chromatin accessibility, TF occupancy, and nucleosome positioning and identified a hierarchy of TFs that govern B cell state, variation, and disease-associated cis-regulatory elements. Perturb-ATAC in primary human epidermal cells revealed three sequential modules of cis-elements that specify keratinocyte fate. Combinatorial deletion of all pairs of these TFs uncovered their epistatic relationships and highlighted genomic co-localization as a basis for synergistic interactions. Thus, Perturb-ATAC is a powerful strategy to dissect gene regulatory networks in development and disease.

Loci identified by a genome-wide association study of carotid artery stenosis in the eMERGE network.

Carotid artery atherosclerotic disease (CAAD) is a risk factor for stroke. We used a genome-wide association (GWAS) approach to discover genetic variants associated with CAAD in participants in the electronic Medical Records and Genomics (eMERGE) Network. We identified adult CAAD cases with unilateral or bilateral carotid artery stenosis and controls without evidence of stenosis from electronic health records at eight eMERGE sites. We performed GWAS with a model adjusting for age, sex, study site, and genetic principal components of ancestry. In eMERGE we found 1793 CAAD cases and 17,958 controls. Two loci reached genome-wide significance, on chr6 in LPA (rs10455872, odds ratio [OR] (95% confidence interval [CI]) = 1.50 (1.30-1.73), p = 2.1 × 10-8 ) and on chr7, an intergenic single nucleotide variant (SNV; rs6952610, OR (95% CI) = 1.25 (1.16-1.36), p = 4.3 × 10-8 ). The chr7 association remained significant in the presence of the LPA SNV as a covariate. The LPA SNV was also associated with coronary heart disease (CHD; 4199 cases and 11,679 controls) in this study (OR (95% CI) = 1.27 (1.13-1.43), p = 5 × 10-5 ) but the chr7 SNV was not (OR (95% CI) = 1.03 (0.97-1.09), p = .37). Both variants replicated in UK Biobank. Elevated lipoprotein(a) concentrations ([Lp(a)]) and LPA variants associated with elevated [Lp(a)] have previously been associated with CAAD and CHD, including rs10455872. With electronic health record phenotypes in eMERGE and UKB, we replicated a previously known association and identified a novel locus associated with CAAD.

An Endoscopic and Histologic Study on Healing of Radiofrequency Ablation Wounds in Patients With Barrett's Esophagus.

INTRODUCTION: Radiofrequency ablation (RFA) of Barrett's esophagus (BE) inflicts a wound spanning 3 epithelial types (stratified squamous, Barrett's metaplasia, gastric epithelium), yet the esophageal injury heals almost completely with squamous epithelium. Knowledge of how this unique wound heals might elucidate mechanisms underlying esophageal metaplasia. We aimed to prospectively and systematically characterize the early endoscopic and histologic features of RFA wound healing. METHODS: Patients with nondysplastic BE had endoscopy with systematic esophageal photographic mapping, biopsy, and volumetric laser endomicroscopy performed before and at 1, 2, and 4 weeks after RFA. RESULTS: Seven patients (6 men; mean age 56.1 ± 10.9 years) completed this study. Squamous re-epithelialization of RFA wounds did not only progress exclusively through squamous cells extending from the proximal wound edge but also progressed through islands of squamous epithelium sprouting throughout the ablated segment. Volumetric laser endomicroscopy revealed significant post-RFA increases in subepithelial glandular structures associated with the squamous islands. In 2 patients, biopsies of such islands revealed newly forming squamous epithelium contiguous with immature-appearing squamous cells arising from esophageal submucosal gland ducts. Subsquamous intestinal metaplasia (SSIM) was found in biopsies at 2 and/or 4 weeks after RFA in 6 of 7 patients. DISCUSSION: RFA wounds in BE are re-epithelialized, not just by squamous cells from the proximal wound margin but by scattered squamous islands in which esophageal submucosal gland duct cells seem to redifferentiate into the squamous progenitors that fuel squamous re-epithelialization. SSIM can be found in most patients during the healing process. We speculate that this SSIM might underlie Barrett's recurrences after apparently successful eradication.

Timing of Resumption of Anticoagulation After Polypectomy and Frequency of Post-procedural Complications: A Post-hoc Analysis.

BACKGROUND: Optimal timing for anticoagulation resumption after polypectomy is unclear. We explored the association between timing of anticoagulation resumption and occurrence of delayed post-polypectomy bleeding (PPB) and thromboembolic (TE) events. METHODS: We performed a post-hoc analysis of patients in an earlier study whose anticoagulants were interrupted for polypectomy. We compared rates of clinically important delayed PPB and TE events in relationship to timing of anticoagulant resumption. Late resumption was defined as > 2 days after polypectomy. RESULTS: Among 437 patients, 351 had early and 86 late resumption. Compared to early resumers, late resumers had greater polypectomy complexity. PPB rate was higher (but not significantly) in the late versus early resumers (2.3% vs. 0.9%, 1.47% greater, 95% CI [- 2.58 to 5.52], p = 0.26). TE events were more frequent in late versus early resumers [0% vs. 1.2% at 30 days, 0% vs. 2.3%, 95% CI 0.3-8, (p = 0.04) at 90 days]. On multivariate analysis, timing of restarting anticoagulation was not a significant predictor of PPB (OR 0.97, 95% CI 0.61-1.44, p = 0.897). Significant predictors were number of polyps ≥ 1 cm (OR 4.14, 95% CI 1.27-13.66, p = 0.014) and use of fulguration (OR 11.43, 95% CI 1.35-80.80, p = 0.014). CONCLUSIONS: Physicians delayed anticoagulation resumption more commonly after complex polypectomies. The timing of restarting anticoagulation was not a significant risk factor for PPB and late resumers had significantly higher rates of TE events within 90 days. Considering the potentially catastrophic consequences of TE events and the generally benign outcome of PPBs, clinicians should be cautious about delaying resumption of anticoagulation after polypectomy.

Integrating regulatory DNA sequence and gene expression to predict genome-wide chromatin accessibility across cellular contexts.

MOTIVATION: Genome-wide profiles of chromatin accessibility and gene expression in diverse cellular contexts are critical to decipher the dynamics of transcriptional regulation. Recently, convolutional neural networks have been used to learn predictive cis-regulatory DNA sequence models of context-specific chromatin accessibility landscapes. However, these context-specific regulatory sequence models cannot generalize predictions across cell types. RESULTS: We introduce multi-modal, residual neural network architectures that integrate cis-regulatory sequence and context-specific expression of trans-regulators to predict genome-wide chromatin accessibility profiles across cellular contexts. We show that the average accessibility of a genomic region across training contexts can be a surprisingly powerful predictor. We leverage this feature and employ novel strategies for training models to enhance genome-wide prediction of shared and context-specific chromatin accessible sites across cell types. We interpret the models to reveal insights into cis- and trans-regulation of chromatin dynamics across 123 diverse cellular contexts. AVAILABILITY AND IMPLEMENTATION: The code is available at https://github.com/kundajelab/ChromDragoNN. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Morbidity and Mortality of Typhoid Intestinal Perforation Among Children in Sub-Saharan Africa 1995-2019: A Scoping Review.

BACKGROUND: Typhoid fever incidence and complications, including intestinal perforation, have declined significantly in high-income countries, with mortality rates <1%. However, an estimated 10.9 million cases still occur annually, most in low- and middle-income countries. With the availability of a new typhoid conjugate vaccine licensed for children and recommended by the World Health Organization, understanding severe complications, including associated mortality rates, is essential to inform country-level decisions on introduction of this vaccine. This scoping review summarizes over 20 years of the literature on typhoid intestinal perforation in sub-Saharan Africa. METHODS: We searched EMBASE, PubMed, Medline, and Cochrane databases for studies reporting mortality rates due to typhoid intestinal perforation in children, under 18 years old, in sub-Saharan Africa published from January 1995 through June 2019. RESULTS: Twenty-four papers from six countries were included. Reported mortality rates ranged from 4.6-75%, with 16 of the 24 studies between 11 and 30%. Thirteen papers included postoperative morbidity rates, ranging from 16-100%. The most documented complications included surgical site infections, intra-abdominal abscesses, and enterocutaneous fistulas. High mortality rates can be attributed to late presentation to tertiary centers, sepsis and electrolyte abnormalities requiring preoperative resuscitation, prolonged perforation-to-surgery interval, and lack of access to critical care or an intensive care unit postoperatively. CONCLUSIONS: Current estimates of mortality related to typhoid intestinal perforation among children in sub-Saharan Africa remain unacceptably high. Prevention of typhoid fever is essential to reduce mortality, with the ultimate goal of a comprehensive approach that utilizes vaccination, improvements in water, sanitation, and hygiene, and greater access to surgical care.

Association of exome sequences with plasma C-reactive protein levels in >9000 participants.

C-reactive protein (CRP) concentration is a heritable systemic marker of inflammation that is associated with cardiovascular disease risk. Genome-wide association studies have identified CRP-associated common variants associated in ∼25 genes. Our aims were to apply exome sequencing to (1) assess whether the candidate loci contain rare coding variants associated with CRP levels and (2) perform an exome-wide search for rare variants in novel genes associated with CRP levels. We exome-sequenced 6050 European-Americans (EAs) and 3109 African-Americans (AAs) from the NHLBI-ESP and the CHARGE consortia, and performed association tests of sequence data with measured CRP levels. In single-variant tests across candidate loci, a novel rare (minor allele frequency = 0.16%) CRP-coding variant (rs77832441-A; p.Thr59Met) was associated with 53% lower mean CRP levels (P = 2.9 × 10(-6)). We replicated the association of rs77832441 in an exome array analysis of 11 414 EAs (P = 3.0 × 10(-15)). Despite a strong effect on CRP levels, rs77832441 was not associated with inflammation-related phenotypes including coronary heart disease. We also found evidence for an AA-specific association of APOE-ε2 rs7214 with higher CRP levels. At the exome-wide significance level (P < 5.0 × 10(-8)), we confirmed associations for reported common variants of HNF1A, CRP, IL6R and TOMM40-APOE. In gene-based tests, a burden of rare/lower frequency variation in CRP in EAs (P ≤ 6.8 × 10(-4)) and in retinoic acid receptor-related orphan receptor α (RORA) in AAs (P = 1.7 × 10(-3)) were associated with CRP levels at the candidate gene level (P < 2.0 × 10(-3)). This inquiry did not elucidate novel genes, but instead demonstrated that variants distributed across the allele frequency spectrum within candidate genes contribute to CRP levels.

Actionable, pathogenic incidental findings in 1,000 participants' exomes.

The incorporation of genomics into medicine is stimulating interest on the return of incidental findings (IFs) from exome and genome sequencing. However, no large-scale study has yet estimated the number of expected actionable findings per individual; therefore, we classified actionable pathogenic single-nucleotide variants in 500 European- and 500 African-descent participants randomly selected from the National Heart, Lung, and Blood Institute Exome Sequencing Project. The 1,000 individuals were screened for variants in 114 genes selected by an expert panel for their association with medically actionable genetic conditions possibly undiagnosed in adults. Among the 1,000 participants, 585 instances of 239 unique variants were identified as disease causing in the Human Gene Mutation Database (HGMD). The primary literature supporting the variants' pathogenicity was reviewed. Of the identified IFs, only 16 unique autosomal-dominant variants in 17 individuals were assessed to be pathogenic or likely pathogenic, and one participant had two pathogenic variants for an autosomal-recessive disease. Furthermore, one pathogenic and four likely pathogenic variants not listed as disease causing in HGMD were identified. These data can provide an estimate of the frequency (∼3.4% for European descent and ∼1.2% for African descent) of the high-penetrance actionable pathogenic or likely pathogenic variants in adults. The 23 participants with pathogenic or likely pathogenic variants were disproportionately of European (17) versus African (6) descent. The process of classifying these variants underscores the need for a more comprehensive and diverse centralized resource to provide curated information on pathogenicity for clinical use to minimize health disparities in genomic medicine.

The relationship between diastolic blood pressure and coronary artery calcification is dependent on single nucleotide polymorphisms on chromosome 9p21.3.

BACKGROUND: Single nucleotide polymorphisms (SNPs) within the 9p21.3 genomic region have been consistently associated with coronary heart disease (CHD), myocardial infarction, and quantity of coronary artery calcification (CAC), a marker of subclinical atherosclerosis. Prior studies have established an association between blood pressure measures and CAC. To examine mechanisms by which the 9p21.3 genomic region may influence CHD risk, we investigated whether SNPs in 9p21.3 modified associations between blood pressure and CAC quantity. METHODS: As part of the Genetic Epidemiology Network of Arteriopathy (GENOA) Study, 974 participants underwent non-invasive computed tomography (CT) to measure CAC quantity. Linear mixed effects models were used to investigate whether seven SNPs in the 9p21.3 region modified the association between blood pressure levels and CAC quantity. Four SNPs of at least marginal significance in GENOA for a SNP-by-diastolic blood pressure (DBP) interaction were then tested for replication in the Framingham Heart Study's Offspring Cohort (N = 1,140). RESULTS: We found replicated evidence that one SNP, rs2069416, in CDKN2B-AS1, significantly modified the association between DBP and CAC quantity (combined P = 0.0065; Bonferroni-corrected combined P = 0.0455). CONCLUSIONS: Our results represent a novel finding that the relationship between DBP and CAC is dependent on genetic variation in the 9p21.3 region. Thus, variation in 9p21.3 may not only be an independent genetic risk factor for CHD, but also may modify the association between DBP levels and the extent of subclinical coronary atherosclerosis.

Novel gene-by-environment interactions: APOB and NPC1L1 variants affect the relationship between dietary and total plasma cholesterol.

Cardiovascular disease (CVD) is the leading cause of death in developed countries. Plasma cholesterol level is a key risk factor in CVD pathogenesis. Genetic and dietary variation both influence plasma cholesterol; however, little is known about dietary interactions with genetic variants influencing the absorption and transport of dietary cholesterol. We sought to determine whether gut expressed variants predicting plasma cholesterol differentially affected the relationship between dietary and plasma cholesterol levels in 1,128 subjects (772/356 in the discovery/replication cohorts, respectively). Four single nucleotide polymorphisms (SNPs) within three genes (APOB, CETP, and NPC1L1) were significantly associated with plasma cholesterol in the discovery cohort. These were subsequently evaluated for gene-by-environment (GxE) interactions with dietary cholesterol for the prediction of plasma cholesterol, with significant findings tested for replication. Novel GxE interactions were identified and replicated for two variants: rs1042034, an APOB Ser4338Asn missense SNP and rs2072183 (in males only), a synonymous NPC1L1 SNP in linkage disequilibrium with SNPs 5' of NPC1L1. This study identifies the presence of novel GxE and gender interactions implying that differential gut absorption is the basis for the variant associations with plasma cholesterol. These GxE interactions may account for part of the "missing heritability" not accounted for by genetic associations.

Novel common and rare genetic determinants of paraoxonase activity: FTO, SERPINA12, and ITGAL.

HDL-associated paraoxonase 1 (PON1) activity is associated with cardiovascular and other human diseases. As the role of genetic variants outside of the PON gene cluster on PON1 activity is unknown, we sought to identify common and rare variants in such loci. We typed 33,057 variants on the CVD chip in 1,362 subjects to test for their effects on adjusted-PON1 activity. Three novel genes (FTO, ITGAL, and SERPINA12) and the PON gene cluster had SNPs associated with PON1 arylesterase (AREase) activity. These loci were carried forward for rare-variant analysis using Exome chip genotypes in an overlapping subset of 1,051 subjects using sequence kernel association testing. PON1 (P = 2.24 × 10(-4)), PON3 (P = 0.022), FTO (P = 0.019), and SERPINA12 (P = 0.039) had both common and rare variants associated with PON1 AREase. ITGAL variants were associated with PON1 activity when using weighted sequence kernel association testing (SKAT) analysis (P = 2.63 × 10(-3)). When adjusting for the initial common variants, SERPINA12 became marginally significant (P = 0.09), whereas all other findings remained significant (P < 0.05), suggesting independent rare-variant effects. We present novel findings that common and rare variants in FTO, SERPINA12, and ITGAL predict PON1 activity. These results further link PON1 to diabetes and inflammation and may inform the role of HDL in human disease.

An evaluation and adjustment method for natural proximal contacts of crowns using diamond dental strips: a case report.

The best way to adjust proximal contacts of newly fabricated indirect restorations has been a long-standing unresolved issue in dentistry. Excessively tight contacts cause incomplete seating of indirect restorations and intrusion of adjacent teeth, which leads to patient discomfort, hypersensitivity, and recurrent dental caries at the crown margins. When seating indirect restorations, interproximal relief should be restored as it exists in natural dentition. This article presents an innovative method of crown seating using diamond strips. This simple, consistent, method makes it easier for clinicians to provide comfortable and long-lasting restorations with minimal time and effort. Laboratory technicians utilize diamond strips to provide properly fitting indirect restorations that require minimal adjustment upon clinical delivery. Diamond strips also allow for accurate determination of heavier proximal contacts, allowing dentists to adjust the proximal contact properly in the patients' mouths. Clinically, restoring natural proximal contacts is a critical factor to the success of indirect restorations. Using this method standardizes proper proximal contact adjustments of laboratory-fabricated indirect restorations between dental labs and dental offices. The method also helps to limit or eliminate the lingering proximal contact issue between clinicians and laboratory technicians.

ATAC-seq Data Processing.

ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) has gained wide popularity as a fast, straightforward, and efficient way of generating genome-wide maps of open chromatin and guiding identification of active regulatory elements and inference of DNA protein binding locations. Given the ubiquity of this method, uniform and standardized methods for processing and assessing the quality of ATAC-seq datasets are needed. Here, we describe the data processing pipeline used by the ENCODE (Encyclopedia of DNA Elements) consortium to process ATAC-seq data into peak call sets and signal tracks and to assess the quality of these datasets.

Deep Learning on Chromatin Accessibility.

DNA accessibility has been a powerful tool in locating active regulatory elements in a cell type, but dissecting the combinatorial logic within these regulatory elements has been a continued challenge in the field. Deep learning models have been shown to be highly predictive models of regulatory DNA and have led to new biological insights on regulatory syntax and logic. Here, we provide a framework for deep learning in genomics that implements best practices and focuses on ease of use, versatility, and compatibility with existing tools for inference on DNA sequence.

Measurement of proximal contact of single crowns to assess interproximal relief: A pilot study.

Background: It is common for dental technicians to adjust the proximal surface of adjacent teeth on casts when fabricating single crowns. However, whether the accuracy of the proximal contact is affected if this step is eliminated is unclear. Objective: To evaluate the accuracy of the proximal contact of single crowns for mandibular first molars fabricated from four different restorative materials, without adjustment of the proximal surface of the adjacent teeth by the laboratory/dental technician. Methods: This study was in vitro; all the clinical procedures were conducted on a dentoform. The mandibular first molar tooth on the dentoform was prepared using diamond burs and a high speed handpiece. Twenty single crowns were fabricated, five for each group (monolithic zirconia, lithium disilicate, metal ceramic, and cast gold). No proximal surface adjacent to the definitive crowns was adjusted for tight contact in the dental laboratory. Both the qualitative analyses, using dental floss and shimstock, and the quantitative analyses, using a stereo microscope, were performed to evaluate the accuracy of the proximal contact of the restoration with the adjacent teeth. In the quantitative analysis, one-way analysis of variance was used to compare mean values at a significance level of 0.05. Results: In quantitative analysis, the differences between the proximal contact tightness of the four groups was not statistically significant (P = 0.802 for mesial contacts, P = 0.354 for distal contacts). In qualitative analysis, in most crowns, dental floss passed through the contact with tight resistance and only one film of shimstock could be inserted between the adjacent teeth and the restoration. However, one specimen from the cast gold crown had open contact. Conclusions: Even without proximal surface adjustment of the adjacent teeth during the crown fabrication process, adequate proximal contact tightness between the restoration and adjacent teeth could be achieved.

Single-Cell and Spatial Transcriptomic Analysis of Human Skin Delineates Intercellular Communication and Pathogenic Cells.

Epidermal homeostasis is governed by a balance between keratinocyte proliferation and differentiation with contributions from cell-cell interactions, but conserved or divergent mechanisms governing this equilibrium across species and how an imbalance contributes to skin disease are largely undefined. To address these questions, human skin single-cell RNA sequencing and spatial transcriptomics data were integrated and compared with mouse skin data. Human skin cell-type annotation was improved using matched spatial transcriptomics data, highlighting the importance of spatial context in cell-type identity, and spatial transcriptomics refined cellular communication inference. In cross-species analyses, we identified a human spinous keratinocyte subpopulation that exhibited proliferative capacity and a heavy metal processing signature, which was absent in mouse and may account for species differences in epidermal thickness. This human subpopulation was expanded in psoriasis and zinc-deficiency dermatitis, attesting to disease relevance and suggesting a paradigm of subpopulation dysfunction as a hallmark of the disease. To assess additional potential subpopulation drivers of skin diseases, we performed cell-of-origin enrichment analysis within genodermatoses, nominating pathogenic cell subpopulations and their communication pathways, which highlighted multiple potential therapeutic targets. This integrated dataset is encompassed in a publicly available web resource to aid mechanistic and translational studies of normal and diseased skin.

The ENCODE Uniform Analysis Pipelines.

The Encyclopedia of DNA elements (ENCODE) project is a collaborative effort to create a comprehensive catalog of functional elements in the human genome. The current database comprises more than 19000 functional genomics experiments across more than 1000 cell lines and tissues using a wide array of experimental techniques to study the chromatin structure, regulatory and transcriptional landscape of the Homo sapiens and Mus musculus genomes. All experimental data, metadata, and associated computational analyses created by the ENCODE consortium are submitted to the Data Coordination Center (DCC) for validation, tracking, storage, and distribution to community resources and the scientific community. The ENCODE project has engineered and distributed uniform processing pipelines in order to promote data provenance and reproducibility as well as allow interoperability between genomic resources and other consortia. All data files, reference genome versions, software versions, and parameters used by the pipelines are captured and available via the ENCODE Portal. The pipeline code, developed using Docker and Workflow Description Language (WDL; https://openwdl.org/) is publicly available in GitHub, with images available on Dockerhub (https://hub.docker.com), enabling access to a diverse range of biomedical researchers. ENCODE pipelines maintained and used by the DCC can be installed to run on personal computers, local HPC clusters, or in cloud computing environments via Cromwell. Access to the pipelines and data via the cloud allows small labs the ability to use the data or software without access to institutional compute clusters. Standardization of the computational methodologies for analysis and quality control leads to comparable results from different ENCODE collections - a prerequisite for successful integrative analyses.

The ENCODE Uniform Analysis Pipelines.

The Encyclopedia of DNA elements (ENCODE) project is a collaborative effort to create a comprehensive catalog of functional elements in the human genome. The current database comprises more than 19000 functional genomics experiments across more than 1000 cell lines and tissues using a wide array of experimental techniques to study the chromatin structure, regulatory and transcriptional landscape of the Homo sapiens and Mus musculus genomes. All experimental data, metadata, and associated computational analyses created by the ENCODE consortium are submitted to the Data Coordination Center (DCC) for validation, tracking, storage, and distribution to community resources and the scientific community. The ENCODE project has engineered and distributed uniform processing pipelines in order to promote data provenance and reproducibility as well as allow interoperability between genomic resources and other consortia. All data files, reference genome versions, software versions, and parameters used by the pipelines are captured and available via the ENCODE Portal. The pipeline code, developed using Docker and Workflow Description Language (WDL; https://openwdl.org/) is publicly available in GitHub, with images available on Dockerhub (https://hub.docker.com), enabling access to a diverse range of biomedical researchers. ENCODE pipelines maintained and used by the DCC can be installed to run on personal computers, local HPC clusters, or in cloud computing environments via Cromwell. Access to the pipelines and data via the cloud allows small labs the ability to use the data or software without access to institutional compute clusters. Standardization of the computational methodologies for analysis and quality control leads to comparable results from different ENCODE collections - a prerequisite for successful integrative analyses.

Dietary cholesterol increases paraoxonase 1 enzyme activity.

HDL-associated paraoxonase 1 (PON1) activity has been consistently associated with cardiovascular and other diseases. Vitamins C and E intake have previously been positively associated with PON1 in a subset of the Carotid Lesion Epidemiology and Risk (CLEAR) cohort. The goal of this study was to replicate these findings and determine whether other nutrient intake affected PON1 activity. To predict nutrient and mineral intake values, 1,402 subjects completed a standardized food frequency survey of their dietary habits over the past year. Stepwise regression was used to evaluate dietary and covariate effects on PON1 arylesterase activity. Five dietary components, cholesterol (P < 2.0 × 10(-16)), alcohol (P = 8.51 × 10(-8)), vitamin C (P = 7.97 × 10(-5)), iron (P = 0.0026), and folic acid (0.037) were independently predictive of PON1 activity. Dietary cholesterol was positively associated and predicted 5.5% of PON1 activity, second in variance explained. This study presents a novel finding of dietary cholesterol, iron, and folic acid predicting PON1 activity in humans and confirms prior reported associations, including that with vitamin C. Identifying and understanding environmental factors that affect PON1 activity is necessary to understand its role and that of HDL in human disease.