DLA Typing The authors hypothesized that regions of the canine genome associated with the presence of SARDS could be identified in a genome-wide association study (GWAS) using a portion of an Affymetrix® custom canine SNP array. Genomic DNA for whole genome analysis was isolated from EDTA-preserved whole blood using the Puregene kit. Fifteen cases and seventeen control Dachshunds were compared across 404,619 SNPs. Cases and controls were analyzed for population stratification by quantile-quantile (Q-Q) plot. PLINK software was used for association analysis.

Genome-Wide Association Study

Sudden acquired retinal degeneration syndrome (SARDS) is a common cause of incurable blindness in dogs. This disease is diagnosed in dogs with a rapid and complete loss of vision combined with a normal appearing ocular fundus and a flatline electroretinogram (ERG; Figure 1). In some cases, affected dogs also exhibit systemic signs like polyuria, polydipsia, polyphagia, lethargy, or weight gain. The cause of this disease is unknown, although an association with endocrinopathies or an autoimmune pathogenesis has been suggested.1 Certain dog breeds are at an increased risk of SARDS, suggesting a heritable component of the disease.2 Discovery of genetic factors predisposing to development of SARDS in Dachshunds (one of the most commonly affected breeds)2 could contribute to a deeper understanding of the disease’s etiology, would assist in identification of at-risk animals, and could potentially lead to the development of treatments.

Background

Evaluation of the MHC class II as a candidate for sudden acquired retinal degeneration syndrome (SARDS) in Dachshunds

Stephanie Stromberg2, Sara Thomasy1, Ariana Marangakis1, Danika Bannasch2 1Department of Surgical and Radiological Sciences; 2Department of Population, Health and Reproduction;

School of Veterinary Medicine, University of California, Davis, Davis, CA, USA

1. Komáromy AM, Abrams KL, Heckenlively JR, et al. Sudden acquired retinal degeneration syndrome (SARDS) – a review and proposed strategies toward a better understanding of pathogenesis, early diagnosis, and therapy. Veterinary Ophthalmology 2015; doi: 10.1111/vop.12291. 2. Auten CR, Thomasy SM, Maggs DJ. Risk factors for sudden acquired retinal degeneration: 151 critically diagnosed cases within a reference population (abstract). Veterinary Ophthalmology 2015; 46: 40. 3. Kennedy LJ, Davison LJ, Barnes A, et al. Identification of susceptibility and protective major histocompatibility complex haplotypes in canine diabetes mellitus. Tissue Antigens 2006a; 68: 467–476. 4. Kennedy LJ, Barnes A, Short A, et al. Canine DLA diversity: 1. New alleles and haplotypes. Tissue Antigens 2007a; 69 (Suppl 1):272–288. 5. [Dachshund]. (n.d.). Retrieved August 23, 2017, from http://dogtime.com/dog-breeds/dachshund#/slide/1

Figure 1. Electroretinograms (ERG) tracings from a normal control dog (blue) and a dog affected with SARDS (red). In the tracing from the normal dog, note the characteristic biphasic waveform consisting of an initial negative deflection (a), which is generated by the photoreceptors, followed by a positive deflection (b) which reflects inner retinal function. In comparison, the ERG tracing of a SARDS-affected dog is described as a "flat-line", due to the absence of detectable a or b waves.

Selected References

Acknowledgements This study was supported by the UC Davis Center for Companion Animal Health, the Vision for Animals Foundation, NIH grants K08 EY021142; P30 EY12576; and T335OD012199.

Based on the results of the GWAS, the authors hypothesized that an association could be identified between specific dog leukocyte antigen (DLA) class II haplotypes and sudden acquired retinal degeneration syndrome (SARDS) in the Dachshund using direct sequencing of genomic DNA from each of the DLA class II genes: DLA-DRB1, DLA-DQA1 and DLA-DQB1. Genomic DNA from 36 case and 68 control Dachshunds was sequenced using locus-specific intronic primers, according to Kennedy et al. (2006a).3 Allele determination was based on annotated sequences deposited in GenBank, beginning with the identification of the alleles present in homozygous individuals, then moving to those of heterozygotes. Haplotype frequencies in cases and controls were compared by Fisher’s exact test. ORs were calculated using a 2 × 2 contingency table.

Figure 2. Manhattan plot comparing 404,619 SNPs of 15 affected and 17 control Dachshunds. Genomic inflation factor = 1. Red line: Bonferroni corrected p value for multiple testings (p = 6.9). Arrow: A preliminary region of association was found on Chromosome 12 at around 2 -3 Mb, although the association did not have genome-wide significance.

A preliminary region of association was found on Chromosome 12 at the approximate location of the DLA class II genes (although the association did not have genome-wide significance). The genes in this region encode for MHC II, an extremely important molecule of the immune system. If the association between MHC II and SARDS is found to be true, it could be a step toward understanding the etiology of the disease.

Figure 3. Expanded view of the region of association on Chromosome 12, overlapping the MHC class II. Stars: approximate loci of the DLA class II genes: DLA-DRB1 (bp 2151409 - 2164564), DLA-DQA1 (bp 2221181 - 2227600) and DLA-DQB1 (bp 2244820 - 2250733).

Allele Cases Case Freq Controls Control Freq p-value OR DRB1*09401 13/60 0.22 7/118 0.06 0.0026 4.39 DQA1*00901 7/68 0.10 36/130 0.28 0.0060 0.30 DQB1*00101 7/64 0.11 41/136 0.30 0.0041 0.28

Table 1. Alleles of DLA-DRB1, DLA-DQA1 and DLA-DQB1 found to be significantly associated with the diagnosis of SARDS (preliminary data).

0 1 2 3 4

1

2

0

3

4

-log1

0(P)

Megabase (Mb)

Discussion

Three alleles were found to be significantly associated with the diagnosis of SARDS with preliminary data (Table 1). Among cases, DRB1*09401 was the second-most common allele of DLA-DRB1. Among controls, DQA1*00901 was the second-most common allele of DLA-DQA1, and DQB1*00101 was the most common allele of DLA-DQB1.

The DLA is a highly polymorphic region of the canine genome in which associations are rarely found on GWAS, so the presence of even a weak association between the DLA and SARDS is worth consideration. The presence of both protective and risk alleles in preliminary DLA typing data supports this association, and could be used as evidence in support of an autoimmune etiology of SARDS.

Chromosome

-log1

0(P)

Class I Class II Class III