Gene co-expression information can be used to evaluate a gene’s function and identify related pathways. To accomplish this for CHRM3 in Ca-HL, a novel bioinformatics pipeline was created to search for genes co-expressed with CHRM3 in an unbiased way genomewide. First, CHRM3 co-expressed genes were identified with COXPRESdb18, a database that provides gene co-expression relationships for animal species, including human, mouse, rat, and others. Next, the top 100 CHRM3 co-expressed genes, as well as CHRM3 itself (total 101 genes), were subjected to enrichment analysis of disease-associated genes using the 2013 version WEB-based GEne SeT AnaLysis Toolkit (WebGestalt)19 with default settings of a minimum number of four genes (out of 101) required for a gene category to be included in the enrichment analysis. Enrichment analysis method “Disease Association Analysis” of WebGestalt was used to test the enrichment of disease-associated genes among these 101 genes. Finally, CHRM3 co-expressed genes that emerged in the top ten enriched gene categories of disease-associated genes were validated for co-expression with CHRM3 using ten different types of human healthy brain tissues (downloaded from Braineac database)17 and the
