Genotyping Array Design and Data Quality Control in the Million Veteran Program.

The Million Veteran Program (MVP), initiated by the Department of Veterans Affairs (VA), aims to collect biosamples with consent from at least one million veterans. Presently, blood samples have been collected from over 800,000 enrolled participants. The size and diversity of the MVP cohort, as well as the availability of extensive VA electronic health records, make it a promising resource for precision medicine. MVP is conducting array-based genotyping to provide a genome-wide scan of the entire cohort, in parallel with whole-genome sequencing, methylation, and other 'omics assays. Here, we present the design and performance of the MVP 1.0 custom Axiom array, which was designed and developed as a single assay to be used across the multi-ethnic MVP cohort. A unified genetic quality-control analysis was developed and conducted on an initial tranche of 485,856 individuals, leading to a high-quality dataset of 459,777 unique individuals. 668,418 genetic markers passed quality control and showed high-quality genotypes not only on common variants but also on rare variants. We confirmed that, with non-European individuals making up nearly 30%, MVP's substantial ancestral diversity surpasses that of other large biobanks. We also demonstrated the quality of the MVP dataset by replicating established genetic associations with height in European Americans and African Americans ancestries. This current dataset has been made available to approved MVP researchers for genome-wide association studies and other downstream analyses. Further data releases will be available for analysis as recruitment at the VA continues and the cohort expands both in size and diversity.

Uptake Kinetics and Trophic Transfer of Tungsten from Cabbage to a Herbivorous Animal Model.

This investigation builds on previous studies on military-relevant tungsten (W) to more thoroughly explore environmental pathways and bioaccumulation kinetics during direct soil exposure versus trophic transfer and elucidate its relative accumulation and speciation in different snail organs. The modeled steady-state concentration and bioaccumulation factor (BAF) of W from soil into cabbage were 302 mg/kg and 0.55, respectively. Steady-state concentrations (34 mg/kg) and BAF values (0.05) obtained for the snail directly exposed to contaminated soil were lower than trophic transfer by consumption of W-contaminated cabbage (tissue concentration of 86 mg/kg; BAF of 0.36). Thus, consumption of contaminated food is the most important pathway for W mobility in this food chain. The highest concentrations of W compartmentalization were in the snail's hepatopancreas based on wet chemistry and synchrotron-based investigations. Chemical speciation via inductively couple plasma mass spectrometry showed a higher degree of polytungstate partitioning in the hepatopancreas relative to the rest of the body. Based on synchrotron analysis, W was incorporated into the shell matrix during exposure, particularly during the regeneration of damaged shell. This offers the potential for application of the shell as a longer-term biomonitoring and forensics tool for historic exposure.

Multi-ancestry GWAS meta-analyses of lung cancer reveal susceptibility loci and elucidate smoking-independent genetic risk.

Lung cancer remains the leading cause of cancer mortality, despite declining smoking rates. Previous lung cancer GWAS have identified numerous loci, but separating the genetic risks of lung cancer and smoking behavioral susceptibility remains challenging. Here, we perform multi-ancestry GWAS meta-analyses of lung cancer using the Million Veteran Program cohort (approximately 95% male cases) and a previous study of European-ancestry individuals, jointly comprising 42,102 cases and 181,270 controls, followed by replication in an independent cohort of 19,404 cases and 17,378 controls. We then carry out conditional meta-analyses on cigarettes per day and identify two novel, replicated loci, including the 19p13.11 pleiotropic cancer locus in squamous cell lung carcinoma. Overall, we report twelve novel risk loci for overall lung cancer, lung adenocarcinoma, and squamous cell lung carcinoma, nine of which are externally replicated. Finally, we perform PheWAS on polygenic risk scores for lung cancer, with and without conditioning on smoking. The unconditioned lung cancer polygenic risk score is associated with smoking status in controls, illustrating a reduced predictive utility in non-smokers. Additionally, our polygenic risk score demonstrates smoking-independent pleiotropy of lung cancer risk across neoplasms and metabolic traits.

Pharmacogenetic allele variant frequencies: An analysis of the VA's Million Veteran Program (MVP) as a representation of the diversity in US population.

We present allele frequencies of pharmacogenomics relevant variants across multiple ancestry in a sample representative of the US population. We analyzed 658,582 individuals with genotype data and extracted pharmacogenomics relevant single nucleotide variant (SNV) alleles, human leukocyte antigens (HLA) 4-digit alleles and an important copy number variant (CNV), the full deletion/duplication of CYP2D6. We compiled distinct allele frequency tables for European, African American, Hispanic, and Asian ancestry individuals. In addition, we compiled allele frequencies based on local ancestry reconstruction in the African-American (2-way deconvolution) and Hispanic (3-way deconvolution) cohorts.

Leveraging the Million Veteran Program Infrastructure and Data for a Rapid Research Response to COVID-19.

Background: The Veterans Health Administration Office of Research and Development (ORD) played a key role in the federal government's response to the COVID-19 pandemic. The ORD effectively leveraged existing resources to answer questions related to the SARS-CoV-2 virus and COVID-19. Observations: When the COVID-19 pandemic hit in 2020, the Million Veteran Program (MVP), one of the largest genomic cohorts in the world, extended the centralized recruitment and enrollment infrastructure to develop a COVID-19 research volunteer registry to assist enrollment in the vaccine and treatment trials in which the US Department of Veterans Affairs (VA) participated. In addition, the MVP allowed for new data collection and a large genomic cohort to understand host contributions to COVID-19. This article describes ways the MVP contributed to the VA's rapid research response to COVID-19. Several host genetic factors believed to play a role in the development and severity of COVID-19 were identified. Furthermore, existing MVP partnerships with other federal agencies, particularly with the Department of Energy, were leveraged to improve understanding and management of COVID-19. Conclusions: A previously established enterprise approach and research infrastructure were essential to the VA's successful and timely COVID-19 research response. This infrastructure not only supported rapid recruitment in vaccine and treatment trials, but also leveraged the unique MVP and VA electronic health record data to drive rapid scientific discovery and inform clinical operations. Extending the models that VA research applied to the federal government at large and establishing centralized resources for shared or federated data analyses across federal agencies will better equip the nation to respond to future public health crises.

Genetic diversity of human RNase 8.

BACKGROUND: Ribonuclease 8 is a member of the RNase A family of secretory ribonucleases; orthologs of this gene have been found only in primate genomes. RNase 8 is a divergent paralog of RNase 7, which is lysine-enriched, highly conserved, has prominent antimicrobial activity, and is expressed in both normal and diseased skin; in contrast, the physiologic function of RNase 8 remains uncertain. Here, we examine the genetic diversity of human RNase 8, a subject of significant interest given the existence of functional pseudogenes (coding sequences that are otherwise intact but with mutations in elements crucial for ribonucleolytic activity) in non-human primate genomes. RESULTS: RNase 8 expression was detected in adult human lung, spleen and testis tissue by quantitative reverse-transcription PCR. Only two single-nucleotide polymorphisms and four unique alleles were identified within the RNase 8 coding sequence; nucleotide sequence diversity (π = 0.00122 ± 0.00009 per site) was unremarkable for a human nuclear gene. We isolated transcripts encoding RNase 8 via rapid amplification of cDNA ends (RACE) and RT-PCR which included a distal potential translational start site followed by sequence encoding an additional 30 amino acids that are conserved in the genomes of several higher primates. The distal translational start site is functional and promotes RNase 8 synthesis in transfected COS-7 cells. CONCLUSIONS: These results suggest that RNase 8 may diverge considerably from typical RNase A family ribonucleases and may likewise exhibit unique function. This finding prompts a reconsideration of what we have previously termed functional pseudogenes, as RNase 8 may be responding to constraints that promote significant functional divergence from the canonical structure and enzymatic activity characteristic of the RNase A family.

Functionally competent eosinophils differentiated ex vivo in high purity from normal mouse bone marrow.

We have devised an ex vivo culture system which generates large numbers of eosinophils at high purity (>90%) from unselected mouse bone marrow progenitors. In response to 4 days of culture with recombinant mouse FLT3-L and recombinant mouse stem cell factor followed by recombinant mouse IL-5 alone thereafter, the resulting bone marrow-derived eosinophils (bmEos) express immunoreactive major basic protein, Siglec F, IL-5R alpha-chain, and transcripts encoding mouse eosinophil peroxidase, CCR3, the IL-3/IL-5/GM-CSF receptor common beta-chain, and the transcription factor GATA-1. BmEos are functionally competent: they undergo chemotaxis toward mouse eotaxin-1 and produce characteristic cytokines, including IFN-gamma, IL-4, MIP-1alpha, and IL-6. The rodent pathogen pneumonia virus of mice replicates in bmEos and elevated levels of IL-6 are detected in supernatants of bmEos cultures in response to active infection. Finally, differentiating bmEos are readily transfected with lentiviral vectors, suggesting a means for rapid production of genetically manipulated cells.

Removal of inorganic mercury by selective extraction and coprecipitation for determination of methylmercury in mercury-contaminated soils by chemical vapor generation inductively coupled plasma mass spectrometry (CVG-ICP-MS).

A procedure is developed for selective extraction of methylmercury (CH3Hg+) from heavily Hg-contaminated soils and sediments for determination by chemical vapor generation inductively coupled plasma mass spectrometry (CVG-ICP-MS). Soils artificially contaminated with 40 µg g-1 inorganic mercury (Hg2+) or methylmercury chloride (CH3HgCl) were agitated by shaking or exposing to ultrasounds in dilute hydrochloric acid (HCl) or nitric acid (HNO3) solutions at room temperature. Extractions in HCl (5 or 10% v/v) resulted in substantial leaching of Hg2+ from soils, whereas 5% (v/v) HNO3 provided selectivity for quantitative extraction of CH3Hg+ with minimum Hg2+ leaching. Agitation with ultrasounds in 5% (v/v) HNO3 for about 3 min was sufficient for extraction of all CH3Hg+ from soils. Coprecipitations with Fe(OH)3, Bi(OH)3 and HgS were investigated for removal of residual Hg2+ in soil extracts. Hydroxide precipitations were not effective. Thiourea or l-cysteine added to soil extracts prior to hydroxide precipitation improved precipitation of Hg2+, but also resulted in removal of CH3Hg+. HgS precipitation was made with dilute ammonium sulfide solution, (NH4)2S. Adding 30 µL of 0.35 mol L-1 (NH4)2S to soil extracts in 5% (v/v) HNO3 resulted in removal of all residual Hg2+ without impacting CH3Hg+ levels. Vapor generation was carried out by reacting Hg2+-free soil extracts with 1% (m/v) NaBH4. No significant interferences were observed from (NH4)2S on the vapor generation from CH3Hg+. The slopes of the calibration curves for CH3HgCl standard solutions in 5% (v/v) HNO3 with and without (NH4)2S were similar. Limits of detection (LOD, 3s method) were around 0.08 µg L-1 for 5% (v/v) HNO3 blanks (n = 10) and 0.10 µg L-1 for 5% (v/v) HNO3 + 0.005 mol L-1 (NH4)2S blanks (n = 10). Percent relative standard deviation (%RSD) for five replicate measurements varied between 3.1% and 6.4% at 1.0 CH3HgCl level. The method is validated by analysis of two certified reference materials (CRM); purely Methylmercury sediment (SQC1238, 10.00 ±â€¯0.291 ng g-1 CH3Hg+) and Hg-contaminated Estuarine sediment (ERM - CC580, 75 ±â€¯4 ng g-1 CH3Hg+ and 132 ±â€¯3 µg g-1 total Hg). CH3Hg+ values for SQC1238 were between 13.0 and 13.2 ng g-1, and 79 and 81 ng g-1 for ERM - CC580. Hg-contaminated soils (57-96 µg g-1 total Hg) collected from the floodplains of Oak Ridge, TN were analyzed for CH3Hg+ using the procedure by CVG-ICPMS. CH3Hg+ levels ranged from 30 to 51 ng g-1 and did not correlate with total Hg levels (R2 = 0.01).

Baseline Characterization and Annual Trends of Body Mass Index for a Mega-Biobank Cohort of US Veterans 2011-2017.

AIM: Million Veteran Program (MVP) is the largest ongoing mega-cohort biobank program in the US with 570,131 enrollees as of May 2017. The primary aim is to describe demographics, military service, and major diseases and comorbidities of the MVP cohort. Our secondary aim is to examine body mass index (BMI), a proxy for general health, among enrollees. MATERIALS AND METHOD: The study population consists of Veterans who actively use the Veterans Health Administration in the US. Data evaluated in this paper combine health information from multiple sources to provide the most comprehensive demographic profile and information on height and weight of MVP enrollees. A standardized cleaning algorithm was used to curate the demographic variables for each participant in MVP. For height and weight, we derived a final data point for each participant to evaluate BMI. STATISTICAL ANALYSIS USED: Multivariable logistic regression was used to compare the differences in BMI categories across enrollment years adjusting for gender, race, and age. P < 0.05 was considered statistically significant. All analyses were conducted using Statistical Analysis System 9.2. RESULTS: The MVP cohort consists of 90.4% of males with an average age of 61.9 years (standard deviation [SD] = 13.9). MVP is the largest multiethnic biobank cohort within the Veteran population with 73.9% White, 19.0% Black, and 6.5% Hispanic. The most common self-reported disease was hypertension (62.6%) for males and depression (47.5%) for females. Mean BMI was 29.7 kg/m2 (SD = 5.8) with 38.2% obese and 42.3% overweight. CONCLUSIONS: Our findings suggest that demographic representation in MVP is similar to the Veterans Health Administration population and contrasts with the overall National Health and Nutrition Examination Survey US population. The prevalence of overweight and obese is high among US Veterans, and future studies will examine the role of BMI and disease risk in the Veteran population.

Impact of population structure on genetic diversity of a potential vaccine target in the canine hookworm (Ancylostoma caninum).

Ancylostoma caninum is a globally distributed canine parasitic nematode. To test whether positive selection, population structure, or both affect genetic variation at the candidate vaccine target Ancylostoma secreted protein 1 (asp-1), we have quantified the genetic variation in A. caninum at asp-1 and a mitochondrial gene, cytochrome oxidase subunit 1 (cox-1), using the statistical population analysis tools found in the SNAP Workbench. The mitochondrial gene cox-1 exhibits moderate diversity within 2 North American samples, comparable to the level of variation observed in other parasitic nematodes. The protein coding portion for the C-terminal half of asp-1 shows similar levels of genetic variation in a Wake County, North Carolina, sample as cox-1. Standard tests of neutrality provide little formal evidence for selection acting on this locus, but haplotype networks for 2 of the exon regions have significantly different topologies, consistent with different evolutionary forces shaping variation at either end of a 1.3-kilobase stretch of sequence. Evidence for gene flow among geographically distinct samples suggests that the mobility of hosts of A. caninum is an important contributing factor to the population structure of the parasite.

Million Veteran Program: A mega-biobank to study genetic influences on health and disease.

OBJECTIVE: To describe the design and ongoing conduct of the Million Veteran Program (MVP), as an observational cohort study and mega-biobank in the Department of Veterans Affairs (VA) health care system. STUDY DESIGN AND SETTING: Data are being collected from participants using questionnaires, the VA electronic health record, and a blood sample for genomic and other testing. Several ongoing projects are linked to MVP, both as peer-reviewed research studies and as activities to help develop an infrastructure for future, broad-based research uses. RESULTS: Formal planning for MVP commenced in 2009; the protocol was approved in 2010, and enrollment began in 2011. As of August 3, 2015, and with a steady state of ≈50 recruiting sites nationwide, N = 397,104 veterans have been enrolled. Among N = 199,348 with currently available genotyping data, most participants (as expected) are male (92.0%) between the ages of 50 and 69 years (55.0%). On the basis of self-reported race, white (77.2%) and African American (13.5%) populations are well represented. CONCLUSIONS: By helping to promote the future integration of genetic testing in health care delivery, including clinical decision making, the MVP is designed to contribute to the development of precision medicine.

Gene expression profiles associated with the transition to parasitism in Ancylostoma caninum larvae.

Ancylostoma caninum is a common canine parasite responsible for anemia and death in infected dogs. Gene expression profiling was used to investigate molecular differences between two different forms of the third larval stage (L3s): infective free-living larvae and in vitro serum-stimulated larvae that mimic the initial stages of parasitism of a host. We developed an A. caninum cDNA microarray consisting of 4191 EST clones, and used it to identify a set of 113 genes that are differentially regulated between infective and parasitic larval stages. Real-time RT-PCR was used to confirm the expression differences of a subset of the genes. Of the genes repressed upon serum stimulation, seven encode members of the 'Ancylostoma secreted protein' ASP family, while another transcript encoding a 24 kDa excretory protein with similarity to ASP was up-regulated in serum-stimulated L3s. This suggests that different members of a protein family that has important implications for the hookworm's parasitic lifestyle are regulated in a complementary manner in response to serum stimulation. Comparison of two strains of A. caninum from North Carolina and Maryland only identified a single gene, one of the members of the ASP family, that was differentially repressed upon serum stimulation.

Characteristics and management of splenic artery aneurysm in liver transplant candidates and recipients.

The incidence of splenic artery aneurysm (SAA) in patients with cirrhosis ranges from 7 per cent to 17 per cent. SAA rupture after liver transplantation (LT) is reported to result in significant morbidity and mortality. We report our experience with SAA in LT candidates. From September 1995 through August 2002, 14 LT candidates were diagnosed with SAA. Twenty SAA occurred in 14 patients with an average diameter of 20 mm. Eleven patients qualified for LT; to date, seven have been transplanted. No intervention for SAA occurred prior to LT. Of the seven patients transplanted, four had SAA identified prior to LT. Three were treated at LT and are alive; the fourth had postoperative splenic artery embolization followed by splenectomy and expired on day 109 from duodenal ulcer complications. Three of seven patients had undiagnosed SAA at LT. One required emergency splenectomy for SAA rupture and is alive at 44 months. The remaining two received no treatment; one suffered a late septic death and one is alive at 15 months. No ruptures occurred in our pre-LT population, suggesting that definitive management can await LT. We recommend that all patients undergo four-phase computed tomography or magnetic resonance angiography (MRA) as part of the LT evaluation and that identified SAA be resected at transplantation.

Eosinophils from lineage-ablated Delta dblGATA bone marrow progenitors: the dblGATA enhancer in the promoter of GATA-1 is not essential for differentiation ex vivo.

A critical role for eosinophils in remodeling of allergic airways was observed in vivo upon disruption of the dblGATA enhancer that regulates expression of GATA-1, which resulted in an eosinophil-deficient phenotype in the DeltadblGATA mouse. We demonstrate here that bone marrow progenitors isolated from DeltadblGATA mice can differentiate into mature eosinophils when subjected to cytokine stimulation ex vivo. Cultured DeltadblGATA eosinophils contain cytoplasmic granules with immunoreactive major basic protein and they express surface Siglec F and transcripts encoding major basic protein, eosinophil peroxidase, and GATA-1, -2, and -3 to an extent indistinguishable from cultured wild-type eosinophils. Fibroblast coculture and bone marrow cross-transplant experiments indicate that the in vivo eosinophil deficit is an intrinsic progenitor defect, and remains unaffected by interactions with stromal cells. Interestingly, and in contrast to those from the wild type, a majority of the GATA-1 transcripts from cultured DeltadblGATA progenitors express a variant GATA-1 transcript that includes a first exon (1E(B)), located approximately 3700 bp downstream to the previously described first exon found in hemopoietic cells (1E(A)) and approximately 42 bp upstream to another variant first exon, 1E(C). These data suggest that cultured progenitors are able to circumvent the effects of the DeltadblGATA ablation by using a second, more proximal, promoter and use this mechanism to generate quantities of GATA-1 that will support eosinophil growth and differentiation.

Predictors of employment after liver transplantation.

Employment after orthotopic liver transplantation (OLT) indicates recipients' physical/psychosocial adjustment. Our aim was to determine clinical, socioeconomic and health-related quality of life parameters influencing employment after OLT. Questionnaire on demographics, medical conditions, alcohol and drug use before/after OLT, and a validated 12-Item Short Form Health Survey (SF-12) were mailed to 126 adult OLT patients. Stepwise logistic regression was conducted to identify best predictors of post-OLT employment. Among non-retirees, 49% were employed after OLT. The predictors of employment were: employment status, income, disability status before OLT and Model of End Stage Liver Disease score. These variables had prediction rate of 82%. Individuals working during the five yr prior to OLT were likely to return to work (p<0.0001), particularly those who held a job for >6 months prior to OLT (p<0.0001), income>$80 000 before OLT compared with <$30 000 (p=0.036). Patients receiving Social Security Insurance (SSI) payment for >or=6 months prior to OLT, were less likely to work (p=0.0005). Severity/duration of liver dysfunction prior to OLT did not correlate with employment. Sense of physical health was poorer in those employed after OLT than in unemployed (p=0.0003). Socioeconomic factors were the most important predictors of post-OLT employment.