Identification of candidate risk gene variations by whole-genome sequence analysis of four rat strains commonly used in inflammation research

Abstract

Background: The DA rat strain is particularly susceptible to the induction of a number of chronic inflammatory diseases, such as models for rheumatoid arthritis and multiple sclerosis. Here we sequenced the genomes of two DA sub-strains and two disease resistant strains, E3 and PVG, previously used together with DA strains in genetically segregating crosses.

Results: The data uncovers genomic variations, such as single nucleotide variations (SNVs) and copy number variations that underlie phenotypic differences between the strains. Comparisons of regional differences between the two DA sub-strains identified 8 genomic regions that discriminate between the strains that together cover 38 Mbp and harbor 302 genes. We analyzed 10 fine-mapped quantitative trait loci and our data implicate strong candidates for genetic variations that mediate their effects. For example we could identify a single SNV candidate in a regulatory region of the gene Il21r, which has been associated to differential expression in both rats and human MS patients. In the APLEC complex we identified two SNVs in a highly conserved region, which could affect the regulation of all APLEC encoded genes and explain the polygenic differential expression seen in the complex. Furthermore, the non-synonymous SNV modifying aa153 of the Ncf1 protein was confirmed as the sole causative factor.

Conclusion: This complete map of genetic differences between the most commonly used rat strains in inflammation research constitutes an important reference in understanding how genetic variations contribute to the traits of importance for inflammatory diseases.

Figure 1

The genomic influence on disease susceptibility. The influence of genomic variations on the development of inflammatory disease by different genomes and MHC haplotypes. The illustration is an adaptation from [14].

Figure 2

Arthritis and EAE regulating QTLs in the DA genome. Chromosome maps illustrating the genomic intervals of inflammatory disease regulating QTLs in the DA rat genome associated with mapping studies in DA/E3 or DA/PVG crosses and congenic strain.

Figure 3

Sequence similarities/differences between DA, E3 and PVG. Similarity is given as the percentage of SNVs shared between combinations of strains out of all SNVs, after excluding 1.01 M positions where at least one strain is heterozygous or has a coverage below 3. The number of SNVs kept for the analysis was 4166021. Total numbers of SNVs are in parenthesis.

Figure 4

The association of SNVs in different gene regions and differential expression or splicing. Association of SNVs in different gene regions and differential expression or splicing, as reported in Gillett et al. [38]. The fractions of genes that have SNVs in i) coding region, ii) UTR or iii) splice sites were measured. a, Genes predicted to be differentially expressed between DA/K and PVG compared to genes with no differential expression in this condition. Three different levels of fold change were used to categorize differentially expressed genes, resulting in 88 genes with fold change (FC) > 2, 214 genes with FC > 1.7, 520 genes with FC > 1.5 and 14140 genes with no differential expression. b, Alternatively spliced genes compared with genes with no evidence of alternative splicing in this condition. Three significance levels were used to categorize significant alternative splicing in genes, resulting in 123 genes in the highest significance category p < 0.00001, 278 (p < 0.01), 613 (p < 0.1) and 13208 genes that were not considered alternatively spliced.

Figure 5

DA/O and DA/K segregating SNV regions. SNVs between DA/O and DA/K plotted on the chromosomes. The dark bands are due to high SNV density in 8 regions that distinguish DA/O and DA/K. Only positions supported by at least 8 reads in both strains were included in the picture.

Figure 6

Snapshots from the UCSC genomic web-browser describing the genomic locations of the identified SNVs within 4 QTLs or specific genes in QTLs (a-d). The SNVs are depicted as red or blue bars in the top of the illustration. The number next to the bar corresponds to the SNPs individual number in the series of all SNPs in the QTL. Blue bars indicate SNVs with conservation score higher than 0.1, in red are SNVs less that 0.1. The mid segment of the snapshot contains the genes in the interval and below is the degree of inter-species conservation illustrated as blue and green bars.