PMC

Genetic associations with wildtype Tyrp1. In BXD RI mice with wildtype Tyrp1, GO enrichment analysis illustrates that the majority of biologic processes to which transcripts correlated with Tyrp1 expression belong include pigmentation, melanin metabolic process, pigment biosynthetic process, melanocyte differentiation and mesenchymal cell development. GO categories reaching statistical significance are indicated with a bold outline and adjusted p values.

Graphic illustration of simple and composite mapping of Tyrp1 expression in whole eyes. A: A significant eQTL is present at the location of Tyrp1 on Chr 4, making it a candidate cis-eQTL. B: Composite interval mapping reveals a suggestive eQTL on chromosome 9 at the location of Myo5a. The blue traces in these interval maps indicate LRS scores across the genome. Horizontal lines mark the transcript-specific significant (LRS 17) and suggestive (LRS 11) thresholds based on results of 1,000 permutations of original trait data.

Partial correlation analysis and modeling of genes with expression in the eye that are modulated by Tyrp1 or Myo5a in the heatmap. A: Summary table showing partial correlation coefficients and corresponding p values for all relationships. B: Composite model showing all Pearson correlation coefficients. Circular lines indicate cis-eQTLs. Linear lines indicate direct regulation of one gene by another. Edges that connect Myo5a directly to other genes/transcripts such as Tyr are labeled with the values of the partial correlation that removes the effects of variation in Tyrp1.

Genetic associations with mutant Tyrp1. In BXD RI mice with mutant Tyrp1, gene ontology enrichment analysis identified two distinct clusters of biologic processes that were correlated with mutant Tyrp1. The first branch included mesenchyme development and neural crest cell development and migration. The second branch included melanin biosynthetic processes and glycoprotein metabolic processes. GO categories reaching statistical significance are indicated with a bold outline and adjusted p values.

Expression levels of Tyrp1 across strains. A: Rank ordered mean of Tyrp1 mRNA expression levels across 67 BXD RI strains, their parental strains and F1 crosses. Values denote normalized relative expression levels on a log2 scale (mean±SEM). The majority of lines with the Tyrp1^b mutation expressed higher levels of Tyrp1 than those with the wildtype allele. F1 and parental strains had intermediate mRNA expression levels. B: western blot showing Tyrp1 protein levels in eyes and irides of parental strains. Tyrp1 protein levels were normalized to Gapdh and the amount of protein in eyes of B6 mice was set to 1. There is no statistical difference in Tyrp1 protein levels between strains with wildtype versus mutant Tyrp1.

Heatmaps of Tyrp1 covariates taken from the GO categories achieving statistical significance in the wildtype data set. A: In the combined data set, both Tyrp1 and Myo5a are cis-eQTLs on chromosome 4 and 9, respectively. The red and blue bands at these same locations indicate which other genes are likely regulated by Tyrp1 and Myo5a. B: In the data set comprised of RI lines with wildtype Tyrp1, the band on chromosome 4 is not present because the expression level of Tyrp1 in these lines does not vary enough to be mapped. However, the band on chromosome 9 remains and is enlarged, suggesting that Myo5a influences the expression of multiple genes in the heatmap. C: In the data set comprised of lines with mutant Tyrp1, Myo5a remains as a cis-eQTL but all other associations are absent. The orange triangles indicate where each gene is located in the genome. Red or blue bars indicate where each gene maps.

Genetic network co-expression graphs. A: Genetic coexpression network generated from genes correlated with wildtype Tyrp1 shows that the expression level of Tyrp1 was directly linked to all nodes in the network with an r value of ≥0.5. Moreover 94% of the genes function in mesenchyme development and/or melanin/pigment production. B: A similar network generated using the same 17 genes and expression levels from the mutant database indicates that the expression of Tyrp1 is correlated with only six genes in the network and the tight cohesive nature of the network is lost. C: Genetic coexpression network generated from genes correlated with mutant Tyrp1 shows that the expression level of mutant Tyrp1 was directly linked to only six nodes at r≥0.7. The remaining associations occurred through intermediate links. Only 8% of the genes in the network function in melanin/pigmentation pathways and only 6% function in mesenchyme development, the two top biologic function categories associated with wildtype Tyrp1. ●=melanin/pigmentation pathway; ■=mesenchyme development; ▲=other biologic function. Each transcript is shown as a node and Pearson correlation coefficient values are indicated as lines between nodes. Bold and thin lines reflect coefficients of 1.0–0.7 and 0.7–0.5 respectively. Red/orange and blue/green colors indicate positive and negative correlation coefficients, respectively.