Prolapse of the third eyelid gland in dogs, characterized by a protuberance of the gland at the free edge of the papillae, results in hypertrophy, hyperplasia, or adenoma, forming a prominent red fleshy mass, often referred to as the cherry eye (Figure 1) (1). The cherry eye may be large, covering a large portion of the cornea, or it may be small and appear only periodically (2). Dogs have three eyelids, two of which are easily visible, and an additional eyelid, the third eyelid, which is usually hidden below the inner corner of the eye (2, 3). The third meibomian gland is located at the base of the T-shaped hyaline cartilage in the roughly triangular conjunctival fold at the corner of the eye and it produces 30–60% of tears (4, 5).

The cause of cherry eyes is currently unknown and is hypothesized to be the laxity of the connective tissue that anchors the third eyelid gland, usually on a genetic basis (6–8). Some researchers have suggested that gland enlargement (lymphoid hyperplasia) is caused by antigenic stimulation due to the exposure of puppies to environmental allergens (5, 7, 9). It can be caused by dysplasia or congenital defects in all current breeds but is more common in brachycephalic dogs, such as American and English Cocker Spaniels, Boston Terriers, Peking Pugs, Beagles, and Bulldogs (8). This is mainly because the facial and orbital appearances of brachycephalic dogs are characterized by a rounded cranial shape and flat orbits, which potentially lead to physiological protrusion of the eyeballs, failing to adequately cover the eyeballs, provide lubrication, and make the cornea less sensitive, leading to a variety of ocular disorders (10). Studies have shown that males have a higher incidence than females and the age of onset is between 3 and 12 months (6, 8, 11). Research has indicated that this disease is most common in dogs that usually eat meat, chicken liver, ham sausage, etc., and are well nourished, which may be due to glandular secretions and sticky glandular excretory ducts caused by poor excretion during the development of complete obstruction (12). Symptomatically, the prolapsed gland grows from small to large slowly in 3–5 days, quickly in 1–2 days, and does not continue to develop after swelling to its maximum size. In most dogs, ocular inflammation is not obvious, and in a few cases, it is mainly caused by ocular discomfort, scratching, or friction from an object. Tumors and inflammation can be ruled out from the limited enlargement of the prolapsed gland and the inflammatory manifestations in the affected eye; however, obstruction of the glandular excretory ducts can explain the above symptoms. The degree of obstruction in the glandular excretory ducts determines the disease course's progression rate, and the gland's histological structure determines the limit to its enlargement (6, 11, 12). Pathophysiologically, the glands are enlarged (6), the follicles are dilated (11), the epithelium of the follicles is detached (11, 12), and the nictitating gland dry (13, 14).

This disease is most common in monocular prolapses, a small number of both eyes prolapse at the same time; there are also eye prolapses after the cure of the prolapse of the other eye, followed by prolapse (11, 13). Dogs with third eyelid glandular hyperplasia are initially enlarged due to infection, and gradually, a red fleshy lump appears at the inner corner of the dog's lower eyelid, which increases in size in a short period (13). Sick dogs have conjunctival redness, tearing, and obvious signs of ocular inflammation and discomfort, as well as a marked increase in ocular discharge. Simultaneously, some sick dogs often scratch their eyes with their front paws; therefore, there is a possibility of mechanical damage to the cornea or aggravation of ocular inflammation, which seriously affects the health and lifespan of domestic dogs. Third eyelid gland prolapse is common in domestic dogs and seriously affects the quality of life of the affected dogs (14).

Cherry eye is usually not painful and does not affect a dog's vision; however, if left untreated, it can cause irritation and inflammation (9). Total glandular excision is commonly used to remove the third meibomian gland or to reposition the gland in domestic dogs affected by cherry eye disease (15). Studies have shown that after the removal of the desmoid gland, there is a decrease in tear production, leading to dry keratoconjunctivitis (KCS) (16). KCS is a chronic inflammatory disease that may result from ductal obstruction and is more likely to occur in females compared to males, mainly due to more intense lymphocytic infiltration of the third oculocutaneous gland, with less secretion (3, 17). KCS recurrence is usually associated with repositioning surgery (6, 18). The current popular treatment is the modified Morgan Pocket technique, which is simpler and less prone to recurrence and complications than total adenomectomy (19, 20).

Although more attention has been paid to the treatment of such diseases in domestic dogs, the genetic background of third eyelid gland prolapse in domestic dogs remains of interest. In this study, we performed a genome-wide association studies (GWAS) to understand the genetic mechanism underlying third eyelid gland prolapse in domestic dogs with ophthalmoplegia.

In this study, GWAS results from 12 dogs with cherry eye compared to 653 brachycephalic dogs showed a total of 52 SNPs exceeding the threshold of significance at the chromosomal level, with 16 SNPs exceeding the threshold of significance at the genomic level (Figure 2A). We genetically annotated SNPs and their upstream and downstream 5Kb regions that exceeded the significance threshold at the chromosome level, and identified a total of 240 genes (Supplementary Table S1), which were analyzed for functional enrichment, and the results showed that these genes were involved in multiple biological functions (Figure 2B). Notably, we identified four SNPs in the CFA3:15627075-15983629 bp region that exceeded the genome-level significance threshold. Chaining analysis of this region revealed that these four SNPs were strongly interlinked (Figure 2C), and gene annotation revealed that this region contained three genes, including KIAA0825, FAM172A, and NR2F1, of which NR2F1 is associated with eye development (22). We found an SNP on CFA22 well above the significance threshold, and chaining analysis of this SNP and its upstream and downstream regions showed that this SNP did not appear to be chained to nearby regions (Figure 2D).

GWAS analysis of 12 cherry-eyed affected dogs vs. 788 brachycephalic and mesocephalic dogs showed 40 SNPs exceeding the threshold of significance at the chromosomal level and 11 SNPs exceeding the significance threshold at the genomic level (Figure 3A). SNPs exceeding the significance threshold at the chromosomal level and their upstream and downstream 5Kb regions were genetically annotated, and a total of 180 genes (Supplementary Table S2) were identified and subjected to functional enrichment analysis, which showed that these genes were involved in multiple biological functions (Figure 3B). Similar to the above results, we found five SNPs in the CFA22:15627075-15983629 bp region that exceeded the significance threshold at the genome level, and chaining analysis was performed on this region, and these five SNPs were found to be strongly chained (Figure 3C).

The results showed that a total of 104 annotated genes were identified in both GWAS analyses, and we further performed functional enrichment analysis on these 104 genes, which showed that there were multiple genes involved in neurodevelopment and other processes. It is worth mentioning that the GO term “GO:0003407” pathway is involved in the neural retinal development pathway (Figure 4), and the genes involved in the high pathway include DIO3 and TTC8 (23–25). Therefore, these 104 genes were analyzed in depth, and 33 were found to be associated with eye development or eye diseases (Supplementary Table S3).

As human companion animals, dogs have the advantages of a large number of naturally occurring genetic diseases with similar body sizes and diets, the same living environment, and a unique population structure that provides high-quality genome-wide reference sequences. Moreover, dog and human genomes have a high degree of similarity (75%), making them ideal animal models for studying spontaneous genetic diseases in humans (26–29). According to the online Human Mendelian Inheritance Database (https://omim.org/home/), as of 2020, ~700 dogs with monogenic diseases or complex traits have been identified, of which at least 230 have known disease-causing mutations and 340 dogs are potential models for human disease (30). The number of spontaneous genetic diseases in other domesticated animals was significantly lower than that in dogs. A large number of dogs with spontaneous genetic diseases can be used as animal models for research, providing more possibilities for studying and treating human genetic diseases (31–33). Third eyelid gland prolapse is a relatively common disease in domesticated animals and has diverse causes (34). A total of 1,802 cases of third eyelid prolapse were identified in 905,543 dogs, with an annual prevalence of 0.20% (35). However, most studies on third eyelid gland prolapse in domestic dogs have focused on the treatment of this disease and the underlying genetic mechanisms have not yet been analyzed. The present study aimed to contribute to the understanding of ocular tumors in humans by analyzing the mechanism of third eyelid gland prolapse.

In this study, we performed GWAS analysis based on 12 dogs with cherry eyes and brachycephalic dogs and identified SNPs. Their upstream and downstream regional gene annotations revealed that some genes are indeed involved in ocular development or are associated with ocular diseases in brachycephalic dogs, NR2F1 may be associated with optic nerve atrophy syndrome. GWAS was also performed on both brachycephalic and mesocephalic dogs, and 33 genes were found to be associated with ocular development or disease in breeds with different cranial structures, including genes associated with diabetic retinopathy, such as SYT3, MYBPC2, and POLD1 (36, 37). These findings are important for exploring the causative genes of third eyelid protrusions in dogs.

In addition to common dog diseases, dogs can also suffer from diseases similar to humans, such as tumors like eye tumors and psychiatric disorders like autism, and studies have shown a strong association between SHANK3 and autism in humans (38, 39). By analyzing the whole genome sequence of domestic dogs, it was found that the structure of the SHANK3 gene in dogs was similar to that of humans. Therefore, the researchers carried out gene editing in dogs, and the results showed that the mutant dogs all showed different degrees of abnormal motor ability, repetitive stereotypy, as well as the presence of severe social abnormalities and cognitive deficits between dogs and dogs, and between dogs and humans, and other clinical symptoms of autism. There are no ideal treatments for fatal human genetic diseases, mainly because of the limited research on the natural history of the disease, insufficient understanding of the correlation between genotype and disease, lack of effective alternative markers, and lag in translational research on effective animal models (40). Therefore, it is necessary to study the natural occurrence and molecular mechanisms of human genetic diseases and evaluate the safety and effectiveness of gene therapy in large animal models (41).

In this study, although we did not fully elucidate the pathogenesis of third eyelid gland prolapse in dogs, this limitation may be attributed to the small sample size or the genetic diversity of the breeds collected. However, we genetically annotated SNPs and their upstream and downstream 5Kb regions associated with canine third eyelid gland ptosis that exceeded the significance threshold at the chromosome level and identified some genes associated with ocular development or disease. This represents a significant step forward in understanding the pathogenesis of third eyelid gland prolapse in dogs.