Fig 1.
Pedigree of dogs used in this study.
Note that this pedigree does not show the entire colony or all siblings of the dogs used in this study, but is presented to illustrate the degree of relatedness between dogs in this study. Circles represent females; squares represent males; open symbols represent non-GRMD (non-dystrophic); blackened symbols represent GRMD (dystrophic); a small circle within a larger circle represents GRMD carrier females; animals listed in more than one position within the pedigree are indicated by a larger circle or square encompassing the primary symbol for the animal, along with a dotted line connecting the multiple positions for that animal in the pedigree. The 9 dogs investigated for this study are listed across the bottom of the pedigree, with “fast” and “slow” labels indicating disease progression speed for GRMD dogs and “control” indicating non-GRMD dogs.
Table 1.
RT-qPCR target and reference gene primers for RNAseq validation.
Table 2.
Summary of RNA sequencing results.
Fig 2.
Principal component and hierarchical analysis for all dogs at both time points.
Principal component 1 (PC1) and Principal component 2 (PC2) were identified by logarithm transformation in DESeq2 at two time points. 75% and 9% variance were explained by PC1 and PC2, respectively. A) shows the principal component analysis for the three groups of dogs: red circles indicate controls, green represents fast-progressing dogs, and blue represents slower-progressing dogs. B) shows the principal component analysis for the two time points: here, red circles represent T1 (age 3 months), and green circles represent T2 (age 6 months). C) is a heatmap showing sample-to-sample distances. Distance was analyzed by logarithm transformation in DESeq2.
Fig 3.
Top 10 DEGs for A) T1: age 3 months, GRMD vs. control; B) T2: age 6 months, GRMD vs. control; C) fast vs. slow progressing GRMD dogs. Green font indicates up-regulated genes; those genes in red font were down-regulated. Venn diagrams on the left of each panel include names of Top 10 DEGs; graphs on the right of each panel indicate numbers of DEGs with functions related to regeneration, metabolism, immune response, both regeneration and metabolism (R,M), both metabolism and immune response (M,I), or both regeneration and immune response (R,I).
Table 3.
Numbers of differentially expressed genes (DEGs) and gene ontology analysis output.
Total numbers of DEGs for the comparisons “Fast vs Control”, “Slow vs Control”, and “Fast vs Slow” are presented in gray rows, with these results broken down by time points below the total numbers (T1 = age 3 months; T2 = age 6 months). For DESeq2, genes with q value < 0.05 were considered as significant. For Gene Ontology, Kyoto Encyclopedia of Genes and Genomes (KEGG), and Panther, genes matched with Canis familaris genome in respective database with q value<0.05 were reported.
Fig 4.
RT-qPCR confirmation of RNAseq results for selected genes at both time points evaluated, and two muscle types, in both fast- and slow-progressing dogs.
The y-axes show the log2 fold change values, while the x-axes provide information about the samples (muscle type and time point). CS = cranial sartorius; VL = vastus lateralis; T1 = age 3 months; T2 = age 6 months. All are normalized to unaffected control dogs and to hPRT using the ΔΔCt method [23]. Error bars show standard error of means.
Fig 5.
Classifications of coding SNVs found in GRMD dogs (but not normal dogs).
Each SNV class is represented by a different color, with relative proportions of each shown as a pie chart.