Introduction

Equine Multiple Congenital Ocular Anomalies (MCOA) syndrome consists of different combinations of clinical symptoms that affect the anterior and posterior segment of the eye. While the syndrome was first described in the Rocky Mountain Horse, where it is highly prevalent, it is not limited to this breed. Additional horse breeds that have been diagnosed with MCOA syndrome include the Icelandic Horse, Shetland Pony, Exmoor Pony, American Miniature Horse, Belgian Draft and Morgan Horse, as well as the Kentucky Mountain Saddle Horse and Mountain Pleasure Horse, both of which are closely related to the Rocky Mountain Horse [1,2,3,4,5,6,7]. MCOA syndrome is inherited as an incompletely dominant trait. Horses with the MCOA phenotype are homozygous for the disease allele and have a wide range of eye anomalies (Figure 1a and b). These include, but are not restricted to, uveal cysts, cornea globosa, iris stromal hypoplasia, abnormal pectinate ligaments, cataracts and iris hypoplasia [1,8,9]. Horses that are heterozygous for the disease causing allele have the less severe Cyst phenotype which mainly consists of cysts that originate from the temporal ciliary body, iris, and/or extend into the temporal, peripheral retina [10,11]. The majority of MCOA syndrome horses also have the valued Silver coat color (Figure 1c). This coat phenotype is characterized by the dilution of black pigment in the hair and is most visible in the mane and tail. The Silver coat color is associated with a missense mutation in exon 11 of premelanosome protein (PMEL) [12,13]. It is not possible to see if a horse carries the Silver mutation on a chestnut background. However, these horses also display the disease phenotype. Selective breeding for the Silver coat color has simultaneously increased the frequency of MCOA syndrome in many breeds as both traits are closely linked on horse chromosome 6 [14].

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Figure 1. Clinical signs of MCOA syndrome and a Silver colored Rocky Mountain Horse.

A) Single or multiloculated cysts originating from the ciliary body, iris and occasionally extending into the retina in the temporal quadrant of the eye is the hallmark of the Cyst phenotype and frequently observed in horses with the MCOA phenotype. However, in the MCOA phenotype, other intraocular anomalies, such as cataracts and mitotic pupils, may block the view of the temporal part of the posterior chamber and peripheral retina. B) The right eye of a Rocky Mountain Horse with ectropion uvea, dyscoria, cataract, and lens subluxation. The granula iridica is hypoplastic, the pupil is misshapen, and circumferential ectropion uvea is present. Nuclear cataract of the nuclearcortical junction is present. Vitreous is present in the anterior chamber between the iris and lens secondary to posterior ventral lens subluxation. C) A shiny white mane and tail, in conjunction with a slightly diluted body color with dapples, is typical of a genetically black Silver colored horse. This horse has also been diagnosed with MCOA. Reprinted from Andersson et al., BMC Genetics 2008, 9:88.

https://doi.org/10.1371/journal.pone.0075639.g001

Mutations in PMEL have previously been shown to regulate hypopigmented phenotypes in mice, chickens, zebrafish, dogs and cattle [15,16,17,18,19]. Only the zebrafish and dog have been reported to have concurrent ocular anomalies, although the clinical presentation in these species is quite different from that observed in the horse [18,20,21]. The zebrafish mutant, fading vision, is characterized by hypopigmented retinal pigment epithelium (RPE) and body melanocytes. Due to defects in RPE, the outer segments of photoreceptors are either strongly reduced in length or are completely absent. Interestingly, the retina of adults shows a partial recovery to normal morphology [18]. No abnormalities in the other structures of the eye were reported. PMEL mutant dogs are called “merles” and typically have blue eyes. The coats of heterozygous dogs are characterized by patches of diluted pigment intermingled with normal hairs, while the coat of homozygous dogs are predominantly white or very pale [19]. One study reported that 54.6% of double merle dogs had mild to severe deafness [22]. To a lesser extent homozygous merle dogs also have an elevated frequency of ocular anomalies including microphthalmia, microcoria, colobomas and increased ocular pressure [20,21]. Merle in the homozygous condition has also been reported to be sublethal due to negative pleiotropic effects on the skeleton, cardiac and reproductive systems [22]. However, not all double merles seem to be affected by theses pleiotropic effects. The reason for this may include modifying genotypes at other loci, strong linkage disequilibrium (LD) between the causative mutation and PMEL (which is positioned in a very gene dense region), or the length variation noted in the SINE element associated with the merle phenotype [22]. Unexpectedly, the SINE element has also been found in a few non-merle dogs [19].

The kinds of mutations identified in PMEL are both numerous and varied, and their phenotypic effects are quite diverse. It is so far not known if any of these mutations completely disrupt protein function. In order to study complete loss of function of PMEL, a mouse line with inactivated Pmel has recently been created [23]. This study showed that Pmel inactivated mice have a minor visible dilution of the coat color and that their melanosomes are spherical, instead of the rod shape present in wild-type animals. Interestingly, no ocular anomalies could be detected in these animals. The very subtle effects of completely disrupting PMEL suggests that PMEL mutant species with more pronounced phenotypic alterations, like the horse, carry dominant negative mutations. In yet another recent study of the biochemical properties of PMEL function, it was shown that mutations in or near the transmembrane domain of PMEL, like the Silver mutation in horse and Dominant white chicken, change the capacity of this region to self-associate [24]. These small in-frame mutations alter fibril formation into more compact structures and this in turn leads to severe pigment loss. Although the effect of the mutation causing the Dominant white phenotype in chicken was proved to be similar to that in the horse, ocular examination of these birds did not identify any ocular anomalies [25].

MCOA syndrome was previously mapped to a 208 kilo-base region on horse chromosome 6 (Andersson et al. 2011b). The region is gene dense and contains several genes that have been proposed to be involved in ocular development or disease. This study aimed at identifying the causative mutation of MCOA syndrome and in doing so, evaluate if PMEL is involved in this eye disease. Further, as the popularity of the Silver coat color is increasing across several horse breeds the results may also have important implications for informed breeding decisions. By next generation DNA sequencing we have managed to exclude all genes, except PMEL, as being causative for both the MCOA syndrome and the Silver coat color in horses.

Results

Discussion

This study establishes that PMEL is causative for MCOA syndrome. We were able to systematically exclude all other candidate genes from a previously identified 208 kb IBD-region using the results of deep sequencing, and in doing so clearly demonstrate the usefulness of this method for finding causative mutations, especially for monogenic disorders where there is a simple relationship between genotype and phenotype. A common outcome of many deep-sequencing projects is the large number of potential candidate mutations identified. In this study, we also show the benefits of well-selected controls. We genotyped control individuals for markers within and flanking each side of the established IBD-region and used this information to identify those that harboured either the ancestral haplotype or that were very close to the disease haplotype for sequencing. By doing so, we dramatically reduced the number of candidate mutations that required validation in additional animals. Further, by using long-range PCRs to build our libraries we were able to cover sequence that may have been missed with other technologies, such as genome gaps and long repeats, which are excluded during sequence capture. More than 90% of our reads mapped to the expected genetic interval with some loss likely due to poor alignment of repetitive sequences in the region.

The previously characterized MCOA syndrome region was 208 kb and contained 15 genes, of which several were potentially important for eye development. In this study we manage to exclude all polymorphic sites, barring two SNPs in PMEL, and propose that the causative mutation is a missense mutation in exon 11 which leads to an arginine to a cysteine (Arg625Cys) change in the second codon of the cytoplasmic region. PMEL is a pigment cell-specific integral membrane protein found in the melanosomes of cells synthesizing primarily the black and brown pigment, eumelanin [27,28]. Subsequent to cleavage, PMEL forms amyloid fibrillar sheets on which melanin is deposited [29]. These sheets form the structural foundation of eumelanosomes and define the organelles maturation stages [30]. The exact function of the fibrillar sheets is not completely understood but they are suggested to have a role in the processing of melanin intermediates, facilitating their polymerization, intracellular or intercellular transfer and/or detoxification [24,28,31]. The first cleavage that PMEL is subjected to breaks the protein into Mα and Mβ fragments. Paradoxically, the Mα fragment forms the actual fibrils important to melanin deposition while the horse missense mutation is within Mβ.

Fortunately, the molecular effects of the two dominant PMEL mutations, Silver in horse and Dominant white in chickens, have recently been studied in detail [24]. This has been accomplished by introducing mutations analogous to these in the context of human PMEL and subsequently evaluating protein function relative to the wild-type form. The horse mutation is positioned very close to the transmembrane domain (TMD) and whereas the wild-type TMD is not able to oligomerize, the mutant form gains this function and can readily self oligomerize. The horse mutation replaces the middle of three consecutive arginines with a cysteine. This new oligomerization ability of the TMD was therefore suggested to be a consequence of decreased electrostatic repulsion between the adjacent basic RRR motifs via the removal of a positive charge [24]. When examining the full-length PMEL, the TMD mutation did not alter protein trafficking or its ability to be processed into amyloidogenic Mα fragments. However, at the end of the cleavage process when a yet unidentified protease cleaves Mβ, there is a small difference in mutant versus wild-type PMEL processing. In wild-type PMEL, Mβ is digested into two different c-terminal fragments (CTF). In contrast, the mutant protein is predominantly digested into just one form, and this seems to be more stable. The consequence of this second finding remains to be evaluated, but may be less drastic than the gain of self oligomerization properties of the TMD [24].

Upon maturation of profibrils into elongated fibrillar sheets, human PMEL containing the analogous horse mutation shows marked differences to the wild-type. In mouse melanocyte cell lines expressing the latter, most stage III and IV melanosomes were densely pigmented. In cells expressing the mutant form, there was less space between the fibrils and the sheets appeared abnormally tightly packed. Exactly how the new TMD oligorization properties affects fibrillar sheet formation remains to be elucidated. Importantly though, and probably because of this arrangement, the organelles seemed to lack pigmentation. This finding suggests that either the fibrils were no longer capable of binding to melanins or melanin production was somehow inhibited. EM analysis revealed that cells expressing mutant PMEL were hypopigmented, harbored fewer pigmented melanosomes and were enriched in early stage melanosomes [24].

These data clearly demonstrate a significant functional consequence of the missense mutation and provide convincing evidence that the coding PMEL mutation is causative for both MCOA syndrome and the Silver phenotype in horses. The obvious further question involves how alterations in the properties of pigmented cells change the whole structure of the eye to a pathologic form. It is not yet known why PMEL mutations give ocular anomalies in some species such as dog, horse and zebrafish, but not in chicken, cow and mouse. This is an interesting topic for further research. Because the relatively mild phenotype in the null mice, all other known PMEL mutations are suggested to be dominant negatives [23].

The silver mutation in mice consists of a single nucleotide insertion that leads to a premature stop codon and a truncated protein, which is missing the last 25 C-terminal amino acids [16,32]. The mutation causes a dilution of eumelanin, which results in the premature graying of the hair due to the loss of follicular melanocytes [33]. In frame insertion/deletions in chicken PMEL are associated with the Dominant white, Dun and Smoky coat colors [17]. Dominant white chickens are completely white and have a nine base pair in frame insertion within the PMEL TMD. These chickens do not present with any ocular anomalies but the homozygotes appeared less pigmented in the outer layers of the choroid than their wild-type counterparts, while the pigment of the retina pigmented epithelium seemed to be unaltered [25]. Interestingly, a second mutation found in a flock of Dominant white chicken, causing the Smokey phenotype, dampens or prevents the accumulation of the pathogenic PMEL isoforms [24]. This mutation behaves like PMEL knockouts and has a much milder phenotype, again suggesting that it is better to have no PMEL at all than the TMD mutant forms. In cattle, dilution of coat color pigment is associated with a missense mutation as well as a three nucleotide, in frame deletion, both residing in the signal peptide of PMEL [15,34]. . The zebrafish mutant, fading vision, exhibits both defects in vision and hypopigmentation and has a point mutation in PMEL, which leads to a truncated protein [18]. The merle patterning of the domestic dog is characterized by patches of diluted pigment and is presumed to be caused by a retrotransposon insertion into intron 10 of PMEL [19]. Some homozygous merle dogs suffer from both auditory and ophthalmologic abnormalities, defects that are similar to those of the human auditory-pigmentation disorder, Waardenburg syndrome [20]. No mutations in human PMEL associated with pigmentation variation have yet been described, but they are likely to exist.

The link between coat color and eye development is not novel and several other known or suggested mutations for coat color have pleiotropic effects, as reviewed in Bellone [35]. In horses these include vision abnormalities (Congenital stationary night blindness in leopard spotted horses), deafness (frame overo patterning and sometimes splashed white patterning), defects in the nervous system (Lethal white foal syndrome in frame overo horses and Lavender foal syndrome) as well as in melanomas (in grey horses) [35,36]. The identification of the MCOA mutation is important not only for resolving the controversial function of PMEL, but it also has practical implications for horse breeders. Molecular testing of the Silver mutation can inform breeding decisions, which is especially meaningful for chestnut or related colors where it is impossible to know if individuals carry the mutation by coat phenotyping alone. Our advice to breeders is to avoid breeding Silver horses to each other. This will essentially eliminate the risk of breeding foals affected by the MCOA phenotype.

Conclusion

The missense mutation g.73665304C>T in the cytoplasmic region of PMEL causes both MCOA syndrome and the Silver coat color. This conclusion is based on the fact that deep sequencing and subsequent analysis eliminated all other possible candidate genes from the IBD-region, and that functional data presented by others showed that the missense mutation causes PMEL function alterations and subsequent changed melanocyte structure.

Materials and Methods

The study was approved by the Ethics Committee for Animal Experiments in Uppsala, Sweden as well as by the Institutional Animal Care and Use Committee at Michigan State University.

Supporting Information

Acknowledgments

We thank Ulla Gustafson for assistance with long-range PCRs and gel purification. Sequencing was performed by the SNP&SEQ Technology Platform in Uppsala. The platform is part of Science for Life Laboratory at Uppsala University.