Genome resequencing and bioinformatic analysis of SNP containing candidate genes in the autoimmune vitiligo Smyth line chicken model

Abstract

Background: The Smyth line (SL) chicken is the only animal model for autoimmune vitiligo that spontaneously displays all clinical and biological manifestations of the human disorder. To understand the genetic components underlying the susceptibility to develop SL vitiligo (SLV), whole genome resequencing analysis was performed in SLV chickens compared with non-vitiliginous parental Brown line (BL) chickens, which maintain a very low incidence rate of vitiligo.

Results: Illumina sequencing technology and reference based assembly on Red Jungle Fowl genome sequences were used. Results of genome resequencing of pooled DNA of each 10 BL and SL chickens reached 5.1x and 7.0x coverage, respectively. The total number of SNPs was 4.8 and 5.5 million in BL and SL genome, respectively. Through a series of filtering processes, a total of ~1 million unique SNPs were found in the SL alone. Eventually of the 156 reliable marker SNPs, which can induce non-synonymous-, frameshift-, nonsense-, and no-start mutations in amino acid sequences in proteins, 139 genes were chosen for further analysis. Of these, 14 randomly chosen SNPs were examined for SNP verification by PCR and Sanger sequencing to detect SNP positions in 20 BL and 70 SL chickens. The results of the analysis of the 14 SNPs clearly showed differential frequencies of nucleotide bases in the SNP positions between BL and SL chickens. Bioinformatic analysis showed that the 156 most reliable marker SNPs included genes involved in dermatological diseases/conditions such as ADAMTS13, ASPM, ATP6V0A2, BRCA2, COL12A1, GRM5, LRP2, OBSCN, PLAU, RNF168, STAB2, and XIRP1. Intermolecular gene network analysis revealed that candidate genes identified in SLV play a role in networks centered on protein kinases (MAPK, ERK1/2, PKC, PRKDC), phosphatase (PPP1CA), ubiquitinylation (UBC) and amyloid production (APP).

Conclusions: Various potential genetic markers showing amino acid changes and potential roles in vitiligo development were identified in the SLV chicken through genome resequencing. The genetic markers and bioinformatic interpretations of amino acid mutations found in SLV chickens may provide insight into the genetic component responsible for the onset and the progression of autoimmune vitiligo and serve as valuable markers to develop diagnostic tools to detect vitiligo susceptibility.

Figure 1

Number of unique SNPs per chromosome found in vitiliginous SL chickens compared to non-vitiliginous BL chickens. Numbers are indicated for bars not clearly visible.

Figure 2

Summary of SNPs in SLV. A) Number of SNPs categorized by chromosomal region in SL chickens. B) Number of SNPs categorized by type of amino acid sequence changes.

Figure 3

Gene network #1. Molecular interactions among important focus molecules are displayed. Gray symbols show the genes found in the list of SNP while white symbols indicate neighboring genes that are functionally associated, but not included, in the gene list of SNP. Symbols for each molecule are presented according to molecular functions and type of interactions.

Figure 4

Gene network #2. Molecular interaction and symbols are the same as the description in Figure 3.

Figure 5

Gene network #3. Molecular interaction and symbols are the same as the description in Figure 3.

Figure 6

Gene network #4. Molecular interaction and symbols are the same as the description in Figure 3.

Figure 7

Gene network #5. Molecular interaction and symbols are the same as the description in Figure 3.

Figure 8

Gene network #6. Molecular interaction and symbols are the same as the description in Figure 3.

Figure 9

Gene network #7. Molecular interaction and symbols are the same as the description in Figure 3.