• Deep-coverage whole genome sequences and blood lipids among 16,324 individuals

      Natarajan, P.; Peloso, G.M.; Zekavat, S.M. (Nature Publishing Group, 2018)
      Large-scale deep-coverage whole-genome sequencing (WGS) is now feasible and offers potential advantages for locus discovery. We perform WGS in 16,324 participants from four ancestries at mean depth >29X and analyze genotypes with four quantitative traits-plasma total cholesterol, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol, and triglycerides. Common variant association yields known loci except for few variants previously poorly imputed. Rare coding variant association yields known Mendelian dyslipidemia genes but rare non-coding variant association detects no signals. A high 2M-SNP LDL-C polygenic score (top 5th percentile) confers similar effect size to a monogenic mutation (~30 mg/dl higher for each); however, among those with severe hypercholesterolemia, 23% have a high polygenic score and only 2% carry a monogenic mutation. At these sample sizes and for these phenotypes, the incremental value of WGS for discovery is limited but WGS permits simultaneous assessment of monogenic and polygenic models to severe hypercholesterolemia. Copyright 2018, The Author(s).
    • Lipid tethering of breast tumor cells reduces cell aggregation during mammosphere formation.

      Bhandary, Lekhana; Bailey, Patrick C; Chang, Katarina T; Underwood, Karen F; Lee, Cornell J; Whipple, Rebecca A; Jewell, Christopher M; Ory, Eleanor; Thompson, Keyata N; Ju, Julia A; et al. (Springer Nature, 2021-02-05)
      Mammosphere assays are widely used in vitro to identify prospective cancer-initiating stem cells that can propagate clonally to form spheres in free-floating conditions. However, the traditional mammosphere assay inevitably introduces cell aggregation that interferes with the measurement of true mammosphere forming efficiency. We developed a method to reduce tumor cell aggregation and increase the probability that the observed mammospheres formed are clonal in origin. Tethering individual tumor cells to lipid anchors prevents cell drift while maintaining free-floating characteristics. This enables real-time monitoring of single tumor cells as they divide to form mammospheres. Monitoring tethered breast cancer cells provided detailed size information that correlates directly to previously published single cell tracking data. We observed that 71% of the Day 7 spheres in lipid-coated wells were between 50 and 150 μm compared to only 37% in traditional low attachment plates. When an equal mixture of MCF7-GFP and MCF7-mCherry cells were seeded, 65% of the mammospheres in lipid-coated wells demonstrated single color expression whereas only 32% were single-colored in low attachment wells. These results indicate that using lipid tethering for mammosphere growth assays can reduce the confounding factor of cell aggregation and increase the formation of clonal mammospheres. © 2021, The Author(s).
    • A lipidome-wide association study of the lipoprotein insulin resistance index

      Bagheri, M.; O'connell, J.; Montasser, M. (Springer Nature, 2020)
      Background The lipoprotein insulin resistance (LPIR) score was shown to predict insulin resistance (IR) and type 2 diabetes (T2D) in healthy adults. However, the molecular basis underlying the LPIR utility for classification remains unclear. Objective To identify small molecule lipids associated with variation in the LPIR score, a weighted index of lipoproteins measured by nuclear magnetic resonance, in the Genetics of Lipid Lowering Drugs and Diet Network (GOLDN) study (n = 980). Methods Linear mixed effects models were used to test the association between the LPIR score and 413 lipid species and their principal component analysis-derived groups. Significant associations were tested for replication with homeostatic model assessment-IR (HOMA-IR), a phenotype correlated with the LPIR score (r = 0.48, p <  0.001), in the Heredity and Phenotype Intervention (HAPI) Heart Study (n = 590). Results In GOLDN, 319 lipids were associated with the LPIR score (false discovery rate-adjusted p-values ranging from 4.59 × 10− 161 to 49.50 × 10− 3). Factors 1 (triglycerides and diglycerides/storage lipids) and 3 (mixed lipids) were positively (β = 0.025, p = 4.52 × 10− 71 and β = 0.021, p = 5.84 × 10− 41, respectively) and factor 2 (phospholipids/non-storage lipids) was inversely (β = − 0.013, p = 2.28 × 10− 18) associated with the LPIR score. These findings were replicated for HOMA-IR in the HAPI Heart Study (β = 0.10, p = 1.21 × 10− 02 for storage, β = − 0.13, p = 3.14 × 10− 04 for non-storage, and β = 0.19, p = 8.40 × 10− 07 for mixed lipids). Conclusions Non-storage lipidomics species show a significant inverse association with the LPIR metabolic dysfunction score and present a promising focus for future therapeutic and prevention studies.
    • Lipids, Apolipoproteins, and Risk of Atherosclerotic Cardiovascular Disease in Persons With CKD

      Bajaj, A.; Xie, D.; Cedillo-Couvert, E. (W.B. Saunders, 2019)
      Rationale & Objective: A large residual risk for atherosclerotic cardiovascular disease (ASCVD) remains in the setting of chronic kidney disease (CKD) despite treatment with statins. We sought to evaluate the associations of lipid and apolipoprotein levels with risk for ASCVD in individuals with CKD. Study Design: Prospective cohort study. Settings & Participants: Adults aged 21 to 74 years with non–dialysis-dependent CKD at baseline enrolled in the Chronic Renal Insufficiency Cohort (CRIC) Study in 7 clinical study centers in the United States. Predictor: Baseline total cholesterol, non–high-density lipoprotein cholesterol (non–HDL-C), very low-density lipoprotein cholesterol (VLDL-C), triglycerides, low-density lipoprotein cholesterol (LDL-C), apolipoprotein B (Apo-B), HDL-C, and apolipoprotein AI (Apo-AI) values stratified into tertiles. Outcome: A composite ASCVD event of myocardial infarction or ischemic stroke. Analytic Approach: Multivariable Cox proportional hazards regression to estimate the risk for ASCVD for each tertile of lipoprotein predictor. Results: Among 3,811 CRIC participants (mean age, 57.7 years; 41.8% white), there were 451 ASCVD events during a median follow-up of 7.9 years. There was increased ASCVD risk among participants with VLDL-C levels in the highest tertile (HR, 1.28; 95% CI, 1.01-1.64), Apo-B levels in the middle tertile (HR, 1.30; 95% CI, 1.03-1.64), HDL-C levels in the middle and lowest tertiles (HRs of 1.40 [95% CI, 1.08-1.83] and 1.77 [95% CI, 1.35-2.33], respectively), and Apo-AI levels in the middle and lowest tertiles (HRs of 1.77 [95% CI, 1.02-1.74] and 1.77 [95% CI, 1.36-2.32], respectively). LDL-C level was not significantly associated with the ASCVD outcome in this population (HR, 1.00 [95% CI, 0.77-1.30] for the highest tertile). Limitations: Associations based on observational data do not permit inferences about causal associations. Conclusions: Higher VLDL-C and Apo-B levels, as well as lower HDL-C and Apo-AI levels, are associated with increased risk for ASCVD. These findings support future investigations into pharmacologic targeting of lipoproteins beyond LDL-C, such as triglyceride-rich lipoproteins, to reduce residual risk for ASCVD among individuals with CKD.
    • Multiancestry Genome-Wide Association Study of Lipid Levels Incorporating Gene-Alcohol Interactions

      Brown, M.R.; Bentley, A.R.; de Vries, P.S. (Oxford University Press, 2019)
      A person’s lipid profile is influenced by genetic variants and alcohol consumption, but the contribution of interactions between these exposures has not been studied. We therefore incorporated gene-alcohol interactions into a multiancestry genome-wide association study of levels of high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and triglycerides. We included 45 studies in stage 1 (genome-wide discovery) and 66 studies in stage 2 (focused follow-up), for a total of 394,584 individuals from 5 ancestry groups. Analyses covered the period July 2014–November 2017. Genetic main effects and interaction effects were jointly assessed by means of a 2–degrees-of-freedom (df) test, and a 1-df test was used to assess the interaction effects alone. Variants at 495 loci were at least suggestively associated (P < 1 × 10−6) with lipid levels in stage 1 and were evaluated in stage 2, followed by combined analyses of stage 1 and stage 2. In the combined analysis of stages 1 and 2, a total of 147 independent loci were associated with lipid levels at P < 5 × 10−8 using 2-df tests, of which 18 were novel. No genome-wide-significant associations were found testing the interaction effect alone. The novel loci included several genes (proprotein convertase subtilisin/kexin type 5 (PCSK5), vascular endothelial growth factor B (VEGFB), and apolipoprotein B mRNA editing enzyme, catalytic polypeptide 1 (APOBEC1) complementation factor (A1CF)) that have a putative role in lipid metabolism on the basis of existing evidence from cellular and experimental models. Copyright The Author(s) 2019.
    • Partial thermal imidization of polyelectrolyte multilayer cell tethering surfaces (TetherChip) enables efficient cell capture and microtentacle fixation for circulating tumor cell analysis

      Ju, J.A.; Lee, C.J.; Thompson, K.N.; Ory, E.C.; Lee, R.M.; Mathias, T.J.; Pratt, S.J.P.; Vitolo, M.I.; Jewell, C.M.; Martin, S.S. (Royal Society of Chemistry, 2020)
      The technical challenges of imaging non-adherent tumor cells pose a critical barrier to understanding tumor cell responses to the non-adherent microenvironments of metastasis, like the bloodstream or lymphatics. In this study, we optimized a microfluidic device (TetherChip) engineered to prevent cell adhesion with an optically-clear, thermal-crosslinked polyelectrolyte multilayer nanosurface and a terminal lipid layer that simultaneously tethers the cell membrane for improved spatial immobilization. Thermal imidization of the TetherChip nanosurface on commercially-available microfluidic slides allows up to 98% of tumor cell capture by the lipid tethers. Importantly, time-lapse microscopy demonstrates that unique microtentacles on non-adherent tumor cells are rapidly destroyed during chemical fixation, but tethering microtentacles to the TetherChip surface efficiently preserves microtentacle structure post-fixation and post-blood isolation. TetherChips remain stable for more than 6 months, enabling shipment to distant sites. The broad retention capability of TetherChips allows comparison of multiple tumor cell types, revealing for the first time that carcinomas beyond breast cancer form microtentacles in suspension. Direct integration of TetherChips into the Vortex VTX-1 CTC isolation instrument shows that live CTCs from blood samples are efficiently captured on TetherChips for rapid fixation and same-day immunofluorescence analysis. Highly efficient and unbiased label-free capture of CTCs on a surface that allows rapid chemical fixation also establishes a streamlined clinical workflow to stabilize patient tumor cell samples and minimize analytical variables. While current studies focus primarily on CTC enumeration, this microfluidic device provides a novel platform for functional phenotype testing in CTCs with the ultimate goal of identifying anti-metastatic, patient-specific therapies.