Enrichment analyses
To test our prediction that males would exhibit increased expression of genes associated with vascularization, we conducted Gene Ontology (GO) enrichment analyses using the R package topGO to identify biological processes that were enriched in genes differentially expressed between males and females (Alexa & Rahnenfuhrer, 2020). We searched for terms related to angiogenesis, the formation of new blood vessels, including the phrase “angio” and removed those not involved in angiogenesis (i.e., “lymphangiogenesis”). We compared the standardized effect of sex for these angiogenesis genes with the standardized effect of sex for all other detectably expressed genes with a Kolmogorov-Smirnov test.
To test our prediction that males would have higher expression of genes associated with estrogen and androgen regulation, we used the Online Predicted Human Interaction Database (OPHID) to assess protein-protein interaction (PPI) networks associated with hormone receptor proteins of interest: ER\(\alpha\), ER\(\beta\), and AR (Brown & Jurisica, 2005). We used ENSEMBL orthology information queried through the R packagebiomaRt (Durinck et al., 2009) to identify one-to-one orthologs in the human genome, then filtered and reindexed our expression matrix to detectably expressed genes in the human genome. We retained a total of 8,255 human genes for analysis. Next we ran a query in OPHID to identify the set of human genes involved in PPI networks with each hormone receptor protein and used biomaRt to convert genes names from UniProt to Ensembl (The UniProt Consortium, 2021). We found the genes associated with PPI networks for ER\(\alpha\), ER\(\beta\), and AR and compared the standardized effect of sex for these genes with the standardized effect of sex for all other detectably expressed genes with human orthologs with a Kolmogorov-Smirnov test.