Linkage disequilibrium mapping in domestic dog breeds narrows the progressive rod-cone degeneration interval and identifies ancestral disease-transmitting chromosome

Abstract

Canine progressive rod-cone degeneration (prcd) is a retinal disease previously mapped to a broad, gene-rich centromeric region of canine chromosome 9. As allelic disorders are present in multiple breeds, we used linkage disequilibrium (LD) to narrow the approximately 6.4-Mb interval candidate region. Multiple dog breeds, each representing genetically isolated populations, were typed for SNPs and other polymorphisms identified from BACs. The candidate region was initially localized to a 1.5-Mb zero recombination interval between growth factor receptor-bound protein 2 (GRB2) and SEC14-like 1 (SEC14L). A fine-scale haplotype of the region was developed, which reduced the LD interval to 106 kb and identified a conserved haplotype of 98 polymorphisms present in all prcd-affected chromosomes from 14 different dog breeds. The findings strongly suggest that a common ancestor transmitted the prcd disease allele to many of the modern dog breeds and demonstrate the power of the LD approach in the canine model.

Figure 1

Retinal micrographs showing non-allelism with prcd for the crosses between Basenji (A, 17.4 wk) or Italian greyhound (B, 17.4 wk) and colony reference prcd-affected dogs; data for the Border collie cross is not illustrated. Retinal photoreceptors are normal. In contrast, crosses between the reference prcd-affected dogs and Australian cattle dog (C, 16.4 wk), Nova Scotia duck tolling retriever (D, 16.1 wk) and Portuguese water dog (E, 26 wk) show disorientation of the photoreceptor outer segments (OS) characteristic of the early stages of prcd. Calibration marker=25μm; RPE=retinal pigment epithelium, IS=inner segment, ONL=outer nuclear layer, OPL=outer plexiform layer, INL=inner nuclear layer.

Figure 2

Schematic representation of the prcd LD interval. Low-pass 3.2X sequence of ∼1.2 Mb from 6 BAC clones from the candidate region was analyzed. Ten affected haplotypes observed in different breeds are illustrated which reduced the LD to ∼106 Kb. Haplotypes 1-4 are common haplotypes found in specific affected breeds: Haplotype 1 in MP, TP, ECS, ACS, LR, PWD and CBR; Haplotype 2 in NSDTR; Haplotype 3 in ACD; Haplotype 4 in AE. Haplotypes 5-10 represent rare recombinant chromosomes observed in ACS (H5), NSDTR (H6), PWD (H7), LR (H8), MP and TP (H9) and TP (H10). Representative SNPs and indels show heterozygosity between the affected chromosomes. The final LD is boxed and contains 98 polymorphisms shared among all affected chromosome, and is represented here by 6 SNPs. Distances and recombination points are not drawn to scale. The 4 markers described in Table 1 (GRB2, AANAT, ST6GalNac2, SEC14L) are in bold letters. For the full data set see Supplementary Table 2A, B. Large black dots in Haplotypes 1 and 2 represent nucleotide deletions.

Figure 3

RNA expression of positional candidate genes (RHBDL6, CYGB, ST6GalNac2, AANAT) in the dog. Expression profile is shown for normal (N) and affected (A) brain and retina, and normal spleen. No difference in expression is observed between affected, and non-affected brain and retina. RHBDL6 shows equal expression in retina and spleen. AANAT and ST6GalNac2 are not expressed in brain or spleen, but are highly expressed in the retina, and have two variants: ∼1.3 kb (major transcript) and ∼3.0 kb for AANAT, and ∼2.2 kb (major transcript) and ∼4.0 kb for ST6GalNac2. CYGB is expressed in brain and retina, but not in spleen, and shows 4 different transcripts. Ribosomal RNA is indicated as 28S and 18S, and β-actin was used as a loading control.

Figure 4

Genetic distance analysis between nine affected chromosomes (both chromosomes from affected ACD, PWD and CBR; single affected chromosomes from and Poodle-NSDTR crossbred and a heterozygous LR). Distances were calculated from 79 SNPs from the prcd candidate region (see Methods). Distances were calculated and clustered using the neighbor-joining method. Confidence in branching is inferred by bootstrap values (B=100). The individual haplotypes separate in one main cluster represented by Poodle, CBR and PWD. Affected chromosomes segregating in the NSDTR and ACD are clearly separated. Note that the PWD and CBR were selected because one chromosome from each was recombinant at SEC14L; the remainder of the haplotype was identical in both. NSDTR=Nova Scotia duck tolling retriever; ACD=Australian cattle dog; PWD=Portuguese water dog; CBR=Chesapeake Bay retriever.