Shared and distinct pathways and networks genetically linked to coronary artery disease between human and mouse

Reviewer #1 (Public Review):

This manuscript represents an elegant bioinformatics approach to addressing causal pathways in vascular and liver tissue related to atherosclerosis/coronary artery disease, including those shared by humans and mice and those that are specific to only one of these species. The authors constructed co-expression networks using bulk transcriptome data from human (aorta, coronary) and mouse (aorta) vascular and liver tissue. They mapped human CAD GWAS data onto these modules, mapped GWAS SNPs to putatively causal genes, identified pathways and modules enriched in CAD GWAS hits, assessed those shared between vascular and liver tissues and between humans and mice, determined key driver genes in CAD-associated supersets, and used mouse single-cell transcriptome data to infer the roles of specific vascular and liver cell types. The overall approach used by the authors is rigorous and provides new insights into potentially causal pathways in vascular tissue and liver involved in atherosclerosis/CAD that are shared between humans and mice as well as those that are species-specific. This approach could be applied to a variety of other common complex conditions.

The conclusions are largely supported by the analyses. Some specific comments:

1. It appears that GWAS SNPs were mapped to genes solely through the use of eQTLs. Current methods involve a number of other complementary approaches to map GWAS SNPs to effector genes/transcripts and there is the thought that eQTLs may not necessarily be the best way to map causal genes.
2. Given the critical causal role of circulating apoB lipoproteins in atherosclerosis in both mice and humans and the central role of the liver in regulating their levels, perhaps the authors could use the 'metabolism of lipids and lipoproteins' network in Fig 3B as a kind of 'positive control' to illustrate the overlap between mice and humans and the driver genes for this network.
3. Is it possible to infer the directionality of effect of key driver genes and pathways from these analyses? For example, ACADM was found to be a KD gene for a human-specific liver CAD superset pathway involving BCAA degradation. Are the authors able to determine or predict the effect of genetically increased expression of ACADM on BCAA metabolism and on CAD? Or the directionality of the effect of the hepatic KD gene OIT3 on hepatic and plasma lipids and atherosclerosis.
4. While likely beyond the scope of this manuscript, the substantial amount of publicly available plasma proteomic and metabolomic data could be incorporated into this multiomic bioinformatic analysis. Many of the pathways involve secreted proteins or metabolites that would further inform the biology and the understanding of how these pathways relate to atherosclerosis.

The findings here will motivate the community of atherosclerosis investigators to pursue additional potential causal genes and pathways through computational and experimental approaches. It will also influence the approach around the use of the mouse model to test specific pathways and therapeutic approaches in atherosclerosis. In addition, the computational approach is robust and could (and likely will) be applied to a variety of other common complex conditions.

Reviewer #2 (Public Review):

Summary:
Mouse models are widely used to determine key molecular mechanisms of atherosclerosis, the underlying pathology that leads to coronary artery disease. The authors use various systems biology approaches, namely co-expression and Bayesian Network analysis, as well as key driver analysis, to identify co-regulated genes and pathways involved in human and mouse atherosclerosis in artery and liver tissues. They identify species-specific and tissue-specific pathways enriched for the genetic association signals obtained in genome-wide association studies of human and mouse cohorts.

Strengths:
The manuscript is well executed with appropriate analysis methods. It also provides a compelling series of results regarding mouse and human atherosclerosis.

Weaknesses:
The manuscript has several weaknesses that should be acknowledged in the discussion. First, there are numerous models of mouse atherosclerosis; however, the HMDP atherosclerosis study uses only one model of mouse atherosclerosis, namely hyperlipidemic mice, due to the transgenic expression of human apolipoprotein E-Leiden (APOE-Leiden) and human cholesteryl ester transfer protein (CETP). Therefore, when drawing general conclusions about mouse pathways not being identified in humans, caution is warranted. Other models of mouse atherosclerosis may be able to capture different aspects of human atherosclerosis. Second, the mouse aorta tissue is atherosclerotic, whereas the atherosclerosis status of the GTEX aorta tissues is not known. Therefore, it is possible that some of the human or mouse-specific gene modules/pathways may be due to the difference in the disease status of the tissues from which the gene expression is obtained. Third, it is unclear how the sex of the mice (all female in the HMDP atherosclerosis study and all male in the baseline HMDP study) and the sex of the human donors affected the results. Did the authors regress out the influence of sex on gene expression in the human data before performing the co-expression and preservation studies? If not, this should be acknowledged. Fourth, some of the results are unexpected, and these should be discussed. For example, the authors identify that the leukocyte transendothelial migration pathway and PDGF signaling pathway are human-specific in their vascular tissue analysis. These pathways have been extensively described in mouse studies. Why do the authors think they identified these pathways as human-specific? Similarly, the authors identified gluconeogenesis and branched-chain amino acid catabolism as human and mouse-shared modules in the vascular tissue. Is there evidence of the involvement of these pathways in atherosclerosis in vascular cells?

Overall, acknowledging these drawbacks and adding points to the discussion will strengthen the manuscript.