Additional evidence supports GRM6 p.Thr178Met as a cause of congenital stationary night blindness in three horse breeds.

Congenital stationary night blindness (CSNB) is an ocular disorder characterized by nyctalopia. An autosomal recessive missense mutation in glutamate metabotropic receptor 6 (GRM6 c.533C>T, p.(Thr178Met)), called CSNB2, was previously identified in one Tennessee Walking Horse and predicted to reduce binding affinity of the neurotransmitter glutamate, impacting the retinal rod ON-bipolar cell signaling pathway. Thus, the first aim was to identify the allele frequency (AF) of CSNB2 in breeds with reported cases of CSNB and breeds closely related to the Tennessee Walking Horse. The second aim was to perform ocular examinations in multiple breeds to confirm the link between genotype and CSNB phenotype. In evaluating 3518 horses from 14 breeds, the CSNB2 allele was identified in nine previously unreported breeds. The estimated AF was highest in pacing Standardbreds (0.17) and lowest in American Quarter Horses (0.0010). Complete ophthalmic examinations and electroretinograms (ERG) were performed on 19 horses from three breeds, including one CSNB2 homozygote from each breed. All three CSNB2/CSNB2 horses had an electronegative ERG waveform under scotopic light conditions consistent with CSNB. The remaining 16 horses (seven CSNB2/N and nine N/N) had normal scotopic ERG results. All horses had normal photopic ERGs. This study provides additional evidence that GRM6 c.533C>T homozygosity is likely causal to CSNB in Tennessee Walking Horses, Standardbreds, and Missouri Fox Trotting Horses. Genetic testing is recommended for breeds with the CSNB2 allele to limit the production of affected horses. This study represents the largest across-breed identification of CSNB in the horse and suggests that this disorder is likely underdiagnosed.

Novel genetic variant associated with globoid cell leukodystrophy in a family of mixed breed dogs.

BACKGROUND: Globoid cell leukodystrophy (GCL) is a fatal autosomal recessive disease caused by variants in the galactosylceramidase (GALC) gene. Two dog breed-specific variants are reported. OBJECTIVES: Characterize the putatively causative GALC variant for GCL in a family of dogs and determine population allele frequency. ANIMALS: Four related mixed-breed puppies with signs of neurologic disease were evaluated. Subsequently, 33 related dogs were tested for genetic markers for parentage and the identified GALC variant. Additional GALC genotyping was performed on 278 banked samples from various breeds. METHODS: The 4 affected puppies had neurological exams and necropsies. DNA was isolated from blood samples. Variants in GALC were identified via Sanger sequencing. Parentage testing was performed using short tandem repeat markers. Prevalence of the GALC variant of interest was investigated in other breeds. RESULTS: GCL was confirmed histopathologically. A novel missense variant in GALC (NC_006590.4:g.58893972G>A) was homozygous in all affected animals (n = 4). A recessive mode of inheritance was confirmed by parentage testing as was variant linkage with the phenotype (LOD = 3.36). Among the related dogs (n = 33), 3 dogs were homozygous and 7 heterozygous. The variant allele was not detected in screening 278 dogs from 5 breeds. The novel variant is either unique to this family or has an extremely low allele frequency in the general population. CONCLUSIONS AND CLINICAL IMPORTANCE: A novel GALC variant was identified that likely explains GCL in this cohort. The identification of multiple causal variants for GCL in dogs is consistent with findings in humans.

Analysis of Genetic Diversity in the American Standardbred Horse Utilizing Short Tandem Repeats and Single Nucleotide Polymorphisms.

American Standardbreds were developed as a harness racing horse breed. The United States Trotting Association closed the studbook in 1973 and implemented a book size cap in 2009. This study aimed to investigate genetic diversity in the American Standardbred after the studbook cap was introduced using short tandem repeats (STRs) and single-nucleotide polymorphisms (SNPs). Sixteen STRs from horses foaled from 2010 to 2015 and their sires and dams (n = 50 621) were utilized to examine allelic richness (Ar), expected heterozygosity (HE), observed heterozygosity (HO), unbiased heterozygosity (HU), inbreeding coefficient (FIS), and fixation index (FST). These analyses found that trotting and pacing sires were less genetically diverse than dams (HEPBonferroni = 0.029 and 6.3 × 10-5, respectively) and their offspring (ArPBonferroni = 0.034 and 6.9 × 10-6, respectively), and pacing offspring were significantly less diverse than their dams (HEPBonferroni = 2 × 10-3). Inbreeding coefficients for trotters (FIS = -0.014) and pacers (FIS = -0.012) suggest that breeding practices have maintained diversity. Moderate levels of genetic differentiation (0.066 < FST < 0.11) were found between pacing and trotting groups. Additionally, 10 of the most prolific trotting sires and their male offspring (n = 84) were genotyped on the 670K Axiom Equine HD Array. HO values higher than HE (P < 0.001), low inbreeding coefficients (mean F = -0.064), and mean FROH = 21% indicate relatively high levels of diversity in this cohort, further supporting the STR data. However, in contrast, HO values were higher for trotting sires (0.41) than their offspring (0.36). This observation warrants further monitoring of diversity over time. These data provide an updated foundation of diversity indices for further, long-term analysis in the breed.

Identification of W13 in the American Miniature Horse and Shetland Pony Populations.

Coat color is a trait of economic significance in horses. Variants in seven genes have been documented to cause white patterning in horses. Of the 34 variants that have been identified in KIT proto-oncogene, receptor tyrosine kinase (KIT), 27 have only been reported in a single individual or family and thus not all are routinely offered for genetic testing. Therefore, to enable proper use of marker-assisted selection, determining breed specificity for these alleles is warranted. Screening 19 unregistered all-white Shetland ponies for 16 white patterning markers identified 14 individuals whose phenotype could not be explained by testing results. In evaluating other known dominant white variants, 14 horses were heterozygous for W13. W13 was previously only reported in two quarter horses and a family of Australian miniature horses. Genotyping known white spotting variants in 30 owner-reported white animals (25 Miniature Horses and five Shetland ponies) identified two additional W13/N American Miniature Horses. The estimated allele frequency of W13 in the American Miniature Horse was 0.0063 (79 N/N, 1 W13/N) and the allele was not detected in a random sample (n = 59) of Shetland ponies. No homozygous W13 individuals were identified and W13/N ponies had a similar all-white coat with pink skin phenotype, regardless of the other white spotting variants present, demonstrating that W13 results in a Mendelian inherited dominant white phenotype and homozygosity is likely lethal. These findings document the presence of W13 in the American Miniature Horse and Shetland pony populations at a low frequency and illustrate the importance of testing for this variant in additional breeds.

Generation of a Biobank From Two Adult Thoroughbred Stallions for the Functional Annotation of Animal Genomes Initiative.

Following the successful creation of a biobank from two adult Thoroughbred mares, this study aimed to recapitulate sample collection in two adult Thoroughbred stallions as part of the Functional Annotation of the Animal Genome (FAANG) initiative. Both stallions underwent thorough physical, lameness, neurologic, and ophthalmic (including electroretinography) examinations prior to humane euthanasia. Epididymal sperm was recovered from both stallions immediately postmortem and cryopreserved. Aseptically collected full thickness skin biopsies were used to isolate, culture and cryopreserve dermal fibroblasts. Serum, plasma, cerebrospinal fluid, urine, and gastrointestinal content from various locations were collected and cryopreserved. Under guidance of a board-certified veterinary anatomic pathologist, 102 representative tissue samples were collected from both horses. Whole tissue samples were flash-frozen and prioritized tissues had nuclei isolated and cryopreserved. Spatially contemporaneous samples of each tissue were submitted for histologic examination. Antemortem and gross pathologic examination revealed mild abnormalities in both stallions. One stallion (ECA_UCD_AH3) had unilateral thoracic limb lameness and bilateral chorioretinal scars. The second stallion (ECA_UCD_AH4) had subtle symmetrical pelvic limb ataxia, symmetrical prostatomegally, and moderate gastrointestinal nematodiasis. DNA from each was whole-genome sequenced and genotyped using the GGP Equine 70K SNP array. The genomic resources and banked biological samples from these animals augments the existing resource available to the equine genomics community. Importantly we may now improve the resolution of tissue-specific gene regulation as affected by sex, as well as add sex-specific tissues and gametes.