Multi-ancestry genome-wide analysis of coronary artery calcification identifies novel loci, functional pathways, and druggable targets
Collaboration for mapping targets from the CHARGE Consortium 1000G GWAS on coronary artery calcification (CAC) to the carotid plaque data in the Athero-Express Biobank Study with Maryam Kavousi, Maxime Bos, Clint L. Miller, and Pat Peyser.
We have bulk RNAseq (n = 635 samples) and single-cell RNAseq data, genome-wide methylation (Illumina 450K) in n ± 600, as well as overlapping genetic data for ±2,100 individuals with extensive histological plaque characterisation. We will address the following questions:
- Gene expression correlated to characteristics of plaques?
- Where target genes expressed, which cell types?
- Are any of the variants associated to plaque characteristics?
We will use the last dataset from the scRNAseq data, including 35 individuals, to project target genes.
For the genetic analyses we will perform regression analyses adjusted for age, sex (where applicable) and principal components. So, we will apply the following model:
model 1: phenotype ~ age + sex + chip-used + PC1 + PC2 + year-of-surgery
phenotypes are:
calcification, codedCalc.binno/minor vs. moderate/heavy stainingcollagen, codedCollagen.binno/minor vs. moderate/heavy stainingfat10, codedFat.bin_10no/<10% fat vs. >10% fatfat40, codedFat.bin_40no/<40% fat vs. >40% fatintraplaque hemorrhage, codedIPH.binno vs. yesmacrophages (CD68), codedmacmean0mean of computer-assisted calculation CD68+ region of interestsmooth muscle cells (alpha-actin), codedsmcmean0mean of computer-assisted calculation SMA+ region of interestintraplaque vessel density (CD34), codedvessel_densitymanually counted CD34+ cells per 3-4 hotspotsmast cells, codedMast_cells_plaquemanually counted mast cell tryptase+ cells (https://academic.oup.com/eurheartj/article/34/48/3699/484981)neutrophils (CD66b), codedneutrophilsmanually counted CD66b+ cells (https://pubmed.ncbi.nlm.nih.gov/20595650/)
Continuous variables were inverse-rank normal transformated, indicated by _rankNorm.
Figure 1: Genotyped individuals in the Athero-Express Biobank Study
For the expression analysis we used carotid plaque-derived bulk RNAseq data and queried it for the gene list. Below a graph showing the overall expression of the genes (not all are in the data) compared to the mean expression of 1,000 randomly picked genes.
Figure 2: Overall expression of target genes in carotid plaques from the Athero-Express Biobank Study
You can load this project in RStudio by opening the file called 'CHARGE_1000G_CAC.Rproj'.
- CHARGE_1000G_CAC.Rmd
- bulkRNAseq.Rmd
- scRNAseq.Rmd
- Parsing_GWASSumStats.Rmd
- RegionalAssociationPlots.Rmd
- PolarMorphism.Rmd
| File | Description | Usage |
|---|---|---|
| README.md | Description of project | Human editable |
| LICENSE | User permissions | Read only |
| .worcs | WORCS metadata YAML | Read only |
| renv.lock | Reproducible R environment | Read only |
| renv | Reproducible R directory | Read only |
| BASELINE | Baseline tables directory | Human editable |
| COLOC_EA_vs_AA | Colocalization results directory | Human editable |
| CredibleSets | Credible sets as calculated and used | Human editable |
| images | Images directory | Human editable |
| OUTPUT | Generic output directory | Human editable |
| PLOTS | Generic plot directory | Human editable |
| PolarMorphism | PolarMorphism results directory | Human editable |
| RACER | Racer output directory | Human editable |
| RACER_AA | Racer African-American (AA) only output directory | Human editable |
| RACER_EA_vs_AA | Racer European-AA output directory | Human editable |
| scripts | Script to process some things | Human editable |
| SNP | SNP analyses scripts and results | Human editable |
| targets | Variant and gene target directory | Human editable |
| "1. CHARGE_1000G_CAC.Rmd" | Setting up work, including baseline, for AE | Human editable |
| "2. bulkRNAseq.Rmd" | Bulk RNAseq analyses in AE (not used) | Human editable |
| "3. scRNAseq.Rmd" | Single-cell RNAseq analyses in AE (not used) | Human editable |
| "4. Parsing_GWASSumStats.Rmd" | Parsing GWAS summary statistics for other analyses | Human editable |
| "5. RegionalAssociationPlots.Rmd" | Regional association plots, mirror and scatter, colocalization plots | Human editable |
| "6. PolarMorphism.Rmd" | Execute genome-wide overlap-analysis using PolarMorphism | Human editable |
| CHARGE_1000G_CAC | Script to process raw data | Human editable |
| CHARGE_1000G_CAC.Rproj | Project file | Loads project |
This project uses the Workflow for Open Reproducible Code in Science (WORCS) to ensure transparency and reproducibility. The workflow is designed to meet the principles of Open Science throughout a research project.
To learn how WORCS helps researchers meet the TOP-guidelines and FAIR principles, read the preprint at https://osf.io/zcvbs/
- To get started with
worcs, see the setup vignette - For detailed information about the steps of the WORCS workflow, see the workflow vignette
Please refer to the vignette on reproducing a WORCS project for step by step advice.
Dr. Sander W. van der Laan is funded through grants from the Netherlands CardioVascular Research Initiative of the Netherlands Heart Foundation (CVON 2011/B019 and CVON 2017-20: Generating the best evidence-based pharmaceutical targets for atherosclerosis [GENIUS I&II]). We are thankful for the support of the ERA-CVD program ‘druggable-MI-targets’ (grant number: 01KL1802), the EU H2020 TO_AITION (grant number: 848146), and the Leducq Fondation ‘PlaqOmics’.
Plaque samples are derived from carotid endarterectomies as part of the Athero-Express Biobank Study which is an ongoing study in the UMC Utrecht.
The framework was based on the WORCS package.
Version: v1.3
Last update: 2023-05-05
Written by: Sander W. van der Laan (s.w.vanderlaan-2[at]umcutrecht.nl).
Description: Script to get some Athero-Express Biobank Study baseline characteristics.
Minimum requirements: R version 3.4.3 (2017-06-30) -- 'Single Candle', Mac OS X El Capitan
Changes log
* v1.3.0 Fixed an issue with LD r2 plotting. Plus cleaned up project.
* v1.2.0 Cleanup of project. Re-initialize and snapshot renv. Also added colocalization between ancestries.
* v1.1.0 Rename. Cleaning up of project (archiving/removing unused code and results).
* v1.0.7 Update to the count data.
* v1.0.6 Update to WORCS framework.
* v1.0.6 Update code and fix coding of plaque vulnerability index (PVI) to work with SNPTEST.
* v1.0.5 Updates to the gene list.
* v1.0.4 Updates to the gene list.
* v1.0.3 Update to the gene list.
* v1.0.2 Updated baseline characteristics information. Update GWASToolKit preparation. Added more candidate SNPs to look at. Updated some variable names.
* v1.0.1 Add an additional phenotype.
* v1.0.0 Initial version. Fixed baseline table, added codes, and results. Created sample-files.
Permission is hereby granted, free of charge, to any person obtaining a copy of this software and associated documentation files (the "Software"), to deal in the Software without restriction, including without limitation the rights to use, copy, modify, merge, publish, distribute, sublicense, and/or sell copies of the Software, and to permit persons to whom the Software is furnished to do so, subject to the following conditions:
The above copyright notice and this permission notice shall be included in all copies or substantial portions of the Software.
THE SOFTWARE IS PROVIDED "AS IS", WITHOUT WARRANTY OF ANY KIND, EXPRESS OR IMPLIED, INCLUDING BUT NOT LIMITED TO THE WARRANTIES OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE AND NONINFRINGEMENT. IN NO EVENT SHALL THE AUTHORS OR COPYRIGHT HOLDERS BE LIABLE FOR ANY CLAIM, DAMAGES OR OTHER LIABILITY, WHETHER IN AN ACTION OF CONTRACT, TORT OR OTHERWISE, ARISING FROM, OUT OF OR IN CONNECTION WITH THE SOFTWARE OR THE USE OR OTHER DEALINGS IN THE SOFTWARE.
Reference: http://opensource.org.