Pigment and Health

The Relationship of Pigment to Health

by Geneva Coats, R.N.

Rare and exotic colors...one of the most attractive and exciting features of our Pomeranians. Variety is the spice of life! Different styles appeal to different breeders. However, there are some important pigment-related health issues to consider.

BREED STANDARDS
Since the inception of purebred dog registries, standards have been established for all breeds. The standard serves to protect a breed from fashion whims. It strives to maintain a breed's traditional "look" and purpose. The standard establishes desirable conformation and temperament characteristics. It provides a goal for breeding practices, and is a guide for breeders and judges. You don't need to understand all the details and theories of genetics, but simply aim to follow the standard when breeding and judging.

But why is a color or pattern so important to the stewards of a breed? Some breed standards disallow excessive white or merle. Many breed standards specify that parti-color dogs have a certain proportion of color to white, or that they must have color extending over both eyes and ears. Most all standards call for full, dark pigmentation of nose, lips, eye rims and pads. Albinism is generally disallowed. Breeders have always been keen scientific observers, even before genetics became an established field. These guidelines were not e stablished for aesthetics' sake, but because certain colors, patterns, and lack of pigmentation were known to be associated with an increase in certain health problems.

There is much documentation and information related to the merle and white spotting patterns, and they will be the focus of this article.

COAT COLOR DEVELOPMENT
The process of coloration and color patterns in dogs starts with embryonic development. The cells which become melanocytes (pigment producing cells) are derived entirely from the neural crest of the embryo. Pigment cells develop from the same cells that give rise to the nervous system. Defects in certain color genes can be associated with defects in the nervous system, as evidenced by vision and hearing problems. If pigment cells are not present, the coat will lack pigment and appear white. Color provides a logical genetic indicator, and explains why it is likely that certain patterned dogs, such as extreme piebalds (>90% white) and merles may be at risk for specific health problems. In studies to date, the coat patterns resulting from genes producing white are significantly associated with deafness. In addition, suppression by "white genes" of pigmentation in the iris of the eye is also associated with deafness. On the other hand, the presence of pigmented, colored patches in the coat is associated with reduced risk of deafness.

WHITE SPOTTING AND HEARING

A coat pattern strongly associated with deafness in dogs is the so-called "S" series-commonly known as white spotting, Irish spotting, or piebalding. Breed examples are the Bull Terrier, Samoyed, Greyhound, Great Pyrenees, Sealyham Terrier, Beagle, Bulldog, Dalmation, English Setter, Papillon and Fox Terrier. Recent studies done by Schmutz have linked the MITF gene to various patterns of white spotting in many breeds. There are different types of insertions at MITF affecting pigmentation in dogs; not all are recessive as has been traditionally believed. Some dogs with white spotting do not have any mutation at MITF, leading us to believe that there are some other genes that also produce white spotting.

The white spotting coat patterns are believed to be related to the path of migration taken by the pigment cells in the embryo. During fetal development, the melanocytes (cells that produce pigment) are concentrated in the neural crest. The neural crest is the area which eventually becomes the brain and spinal cord. From the neural crest, the pigment cells migrate to the peripheral areas of the body, such as the head, legs and tail. However, sometimes the melanocytes don't reach the farthest distal areas of the body. This could be due to the effects of genes, or it might happen because of an environmental factor during development (such as nutrition or infection). Any areas where the pigment cells fail to migrate will remain white. This is why sometimes dogs will have a white spot on the end of the tail, the tips of the toes, or on the chest. If pigment-producing melanocytes fail to reach the inner ear, deafness will result.

The gene insertions at MITF that produce white spotting patterns are believed to suppress expression of melanocytes during embryogenesis and fetal development. In addition to regulating the differentiation of neural-crest derived melanocytes, MITF is also involved in several other developmental processes. It controls the retinal pigment layers in the eye. MITF mutations in mice cause reduced eye size, failure of secondary bone resorption, and early onset of deafness.

White color on the head is often, but not always, associated with deafness due to lack of inner ear pigment. Patched-colored dogs with dark eyes have been selected for in many breeds. These dogs have reduced incidence of hearing impairment when compared to dogs which are almost totally white.

Not all breeds with an extreme piebald pattern have been reported to be affected with deafness, and there is also inherited deafness in several breeds which is totally unrelated to coat color.

Both white spotting and merle are known to sometimes affect hearing. In breeds which have traditionally included the merle color pattern, the predominantly white (double-factored) merle has been disallowed in the show ring, as well as selected against in the whelping box.

WHAT IS MERLE?

“Merle is a coat pattern caused by a dominant mutation in a pigmentation gene called SILV. The mutation (an insertion of repetitive, mobile DNA) causes dilution of the base fur color and often blue eye color. Merle affects all coat colors, but is more apparent in dogs with darker colored coats (e.g., black) than those with lighter-colored coats (e.g., sable). Coat color is determined by another gene. Because of the type of mutation that causes merle, merling is random and characteristics such as the size and number of colored patches and eye color can not be predicted.” Dr Leigh Anne Clark

Merle is a coat pattern, not a color. The merle gene works like other dilution genes, in that it lightens whatever color is already there. Whatever the basic color of the dog-unless he is all white-the merle gene will cause dilution and splotching imposed on the base coat color. The appearance is a speckling or marbling, like when bleach is splashed on your clothes. The pigment cells are affected individually in the fetus. Some are disabled completely, leaving white areas. Some c ells allow partial expression of pigment, leaving merled or dappled areas. Some cells are unaffected, leaving the coat color normal or non-merled. The amount of dappling or merling can vary. The dog can have perhaps just a small dappled spot somewhere, or perhaps just a light eye with a nondappled, normal coat. Or, it might display dappling throughout the coat. The effect is most noticeable on a black coat; where the black is diluted to grey, the color is called "blue merle".
Merle appears at first glance to be dominant, because if a dog carries one gene for the merle factor, he will be affected by it. However, merle is different, because it is incompletely dominant. A single merle gene, inherited from one parent, produces what is known as a "heterozygous" or "single" merle. This single copy of the gene will usually cause the affected dog to display merling somewhere in the coat. This can range from light dappling to extensive dappling. Sometimes the effect on the coat is not evident, and there is no way to tell at maturity that the dog carries the merle gene. Sometimes, blue flecks in the eyes are the the only sign that a dog carries the merle gene.

If two heterozygous merles are bred together, statistically 25% of the offspring will inh erit two merle factor genes, one from each parent. When this happens, we produce what is known as a "double (homozygous) merle". The effect of the gene is then doubly intense. There is so much color dilution that the dog is usually predominantly white, and almost always has impaired hearing or vision-or both. Many double merle dogs do not survive to birth.

In French, the word "merle" means "blackbird", and "le merle blanc" ("The white blackbird") is an expression, meaning something that is impossible or something that cannot exist. This is an admirable goal for the white merle dog!

In Great Danes, merle interacts with another gene called harlequin, which bleaches the gray areas of the coat to white. In the pure homozygous form, harlequin is lethal. In Danes and Aussies, the tweed pattern is a variation on merle which adds a third, intermediate shade to the coat.

HOW MERLE WORKS

Merle is a "transposon", or transposable element; a mobile parasite segment of DNA in the chromosome. Transposons usually originate from viruses. A virus is a particle containing DNA which inserts itself into the host cell, thereby infecting it. If the virus does not kill the host cell, it can leave behind a piece of its genetic material in the DNA of the host cell. When this happens in the sex or germinal cells, the particle can become a part of th e genetic makeup of some of the offspring. This is a way that mutation occurs.

Transposons have also been called "junk DNA or "selfish DNA", as they rarely provide any benefit to the host cell. Transposons are also often referred to as "jumping genes." The transposon can move to different positions in the cell in a "cut and paste" process. In so doing, it may cause mutations, or alter the amount of DNA in the genome.

The merle transposon segment consists of a head, a body and a longer tail segment. During cellular replication of early embryonic development, the tail segment of the merle gene may lose some of its DNA nucleotide particles. Coat color in that area will then be less affected by the gene, or possibly even unaffected, and the underlying true coat color will be expressed. This explains why there are some areas of the coat which are NOT dappled, and other areas which ARE. This may also explain how cryptic merles can be produced.

The decorative variegated Indian corn is produced by transposon elements in the corn gene. Many viruses, such as those responsible for AIDS and feline leukemia, function as transposons. In humans, hereditary diseases such as hemophilia A and B, predisposition to colon polyps and cancer, and Duchenne muscular dystrophy, are all related to inherited mutations produced through the transposon process. Genetic engineers sometimes use transposons to try to intentionally introduce certain genes into an organism.

There are also documented cases (about 3-4% incidence) of homozygous merles producing non-merle offspring. This process is known as "germinal reversion". This is further evidence of the instability of the gene. Not only can you sometimes get non-merle pups (which should not happen according to the rules of Mendelian genetics), but worse things can happen. The "cleft palate syndrome" is a rare and isolated Aussie defect that has been the subject of numerous scientific journal articles and symposia presentations. It is a sex-linked (on the X chromosome) defect, in which females have minor abnormalities, like extra toes, while males die of massive skeletal abnormalities and a cleft palate. This disease started with a homozygous merle bitch that was kept for coat color research.

Researchers have tested merle dogs in many breeds and found that all merle dogs have the same merle gene, inserted at the identical location. If this same gene were inserted at a different color location, it would produce a uniform coat color dilution, similar to other dilution genes, rather than a mottled effect. There was no question in this regard with any merle dog in any breed researchers have tested: merle is found in a wide variety of breeds and it is an old mutation. All merle dogs share common ancestry.

MERLE AND EYESIGHT

There is a complex of eye defects associated with the merle gene. These defects can be superficial in nature, such as a difference in color between the iris of one eye and the other. The example of this is a dog with one brown eye and one blue eye. (Note that this trait is not necessarily indicative of possession of the merle gene, because it can also be found in dogs with extreme white spotting). In addition to superficial indicators there are also major effects, such as absence of the reflective substance which lines the back of the eye, resulting in reduced ability to see in low light. There can be lack of retinal pigment, which directly reduces vision. Small or absent eyeballs, irregular or starburst pup ils, and clefts in the iris can occur, as well as persistent pupillary membrane, strabismus (cross-eyed), lens luxation , and juvenile cataracts.

The exact mechanism by which merle produces most these eye problems is unknown. Eye pigment reduction to blue is presumed due to suppression of pigment in the iris.

THE MERLE LINK TO MICROPHTHALMIA

The term "microphthalmia" means a smaller than normal, tiny eyeball due to a defect in early development. Microphthalmia is a defect very commonly associated with merle; particularly homozygous "double" merle, it even rarely occurs in heterozygous or "single" merles. Complete absence of the eyeball in the eye socket sometimes occurs. This is known as "anophthalmia".

MITF, or "Microphthalmia Transcription Factor", was recently discovered to be the location of genes that affect pigmentation, causing white spotting (otherwise known as the particolor or piebald pattern).

The merle gene is believed to possibly affect eye development through some sort of interaction with MITF. Now, why would microphthalmia occur in association with merle and not just with white coat color in particolor dogs? I've asked several canine geneticists, but have received no answer! This relationship is still a mystery. The exact mechanism by which merle interacts with the white spotting gene and produces the defect of microphthalmia is still waiting to be discovered!

MERLE AND HEARING
Hearing impairment can occur in merles due to a lack of melanin or pigment in the inner ear. Not all merles will develop problems with hearing, it all depends on whether or not the inner ear is pigmented. If the inner ear isn't pigmented, the nerve cells responsible for hearing can't develop as they should. Then, the nerve endings atrophy and die off in the first few weeks of the puppy's life, resulting in partial or total deafness. The deafness is neither dominant nor recessive, but is linked to the merle gene which disrupts pigmentation and secondarily produces deaf dogs.

Canine geneticist Dr. Leigh Anne Clark, now working at Clemson University in South Carolina, has done extensive research on the merle and harlequin coat patterns. Dr. Clark notes: “The biggest factor that I have noticed with deafness in heterozygous merles is whether there are other coat color genes involved - dogs that also have piebald seem to be more likely to have problems.”

White outer ear color is often associated with lack of inner ear pigment and deafness. However, sometimes deafness occurs even with patches of color on the ears. Mild to moderate or unilateral hearing impairment may never be noticed, and is rarely tested for.

ONE COPY OF THE GENE HARMLESS?

The popular belief is that heterozygous merles are completely normal, and that only homozygous merles have problems. Heterozygous merles can have expression of lesser defects of the eye such as clefts in the iris, and a thinning of the retina (similar to what is found with Collie eye anomaly). There are also rare cases of microphthalmia in "single" merles. Deafness can also sometimes occur, if the merle gene prevents pigment deposition in the nerve cells of the inner ear during embryonic development.

Heterozygous "single" merles often have reduced eye pigment which produces the characteristic blue eyes. Interestingly, the amount of white even in homozygous merles does not correlate to severity of eye defects. In a study of the embryonic origin of merle eye defects, Dr. Cynthia Cook, of the University of California , San Francisco , observed that the severity of eye defect and amount of pigment were not related. This is in contrast to hearing impairment in merles; the likelihood of deafness increases with increasing amounts of white in the hair coat.

According to researchers, merle combined with piebald increases the risk of deafness. Future testing of vision and hearing in merle dogs will hopefully paint a clearer picture for us.

GENETIC LINKAGE CAN CAUSE SPREAD OF DEFECTS

Genes on the same chromosome are usually inherited together. This process is known as genetic linkage. However, during cellular division and replication, sometimes chromosomes will randomly break and recombine. Genes that were formerly associated with each other can then become separated and inherited independently. We have already mentioned the Aussie cleft palate syndrome which originated with a merle bitch. Iris coloboma, or missing part of the iris, is being reported in non-merle Aussies. Coloboma appears as a notch at the edge of the pupil, giving the pupil an irregular shape. This sight-altering defect is believed to have been brought into the breed through linkage with the merle factor. This trait is now inherited independently from merle.

It is likely more than coincidence that breeds in which merle is most common are also breeds which are heavily plagued with a variety of eye defects. Yearly CERF testing by a veterinary ophthalmologist can help screen out heritable abnormalities. Further studies in merle dogs are needed to observe the frequency and exact methods by which eye defects are produced.

BREEDING RECOMMENDATIONS FOR MERLE

Dogs with one merle gene are generally unaffected with serious health problems, so these can be avoided by breeding a merle dog only to a non-merle.

Sometimes a dog is genetically carrying the merle factor gene, but there are no outward signs (cryptic or phantom merle). This dog could easily be inadvertently bred to another merle and result in the production of homozygous double merles. For this reason, it is usually recommended to avoid breeding merle to sable, brindle or any predominantly phaeomelanin (orange-red-gold-yellow) coat color.

It is almost universally recommended to NEVER breed two merles together, to avoid producing defective double merles. But, unfortunately, some breeders will intentionally breed merle to merle, hoping to produce a higher percentage of merle offspring. This is a sadly misinformed idea. Others may breed merle to merle in an attempt to produce a double merle sire to use for future breeding. Such a double merle dog (even if vision or hearing impaired) will produce 100% merle offspring. There is a demand for the unusual merle color. Although this method of breeding may be considered unethical, it does happen in many breeds, by uneducated or uncaring breeders.

A merle dog should be bred to a dark colored mate, preferably black or black and tan. A dominant black dog will only produce black offspring half the time, unless he carries two copies of the dominant gene for black. Couple a black with a merle parent, who will produce merle offspring 50% of the time, and the odds just dropped to 25% for producing a blue merle from a black to merle mating...the same odds of producing a sable merle from such a breeding.

Black and tanpoint pattern is recessive and would breed true. If one parent is also merle, you have a 50/50 chance for producing blue merle offspring, and 50/50 tanpoint who are not merle. Recessive black would also breed true, just as black and tan does, but recessive black seems to occur only very rarely in our breed. Also, In our breed, there is a widely carried recessive gene "e" which suppresses all black pigment in the coat. This gene can double up in the offspring to hide the visual appearance of merle.

Here is part of a statement by canine geneticist Sheila Schmutz, written for the Pomeranian standard revision committee in April 2006:

"Yet another gene in the "blue" family that can cause health issues is merle. Merle can not be seen in dogs with an e/e genotype. This e/e genotype occurs commonly in Pomeranians since orange, red and white probably account for the majority of Poms. The problem is that M/M (homozygous merle) dogs are always deaf based on our studies. We recently genotyped 24 mostly white Australian Shepherds and all tested M/M (based on the Clark et al. 2006 PNAS published test) and all were deaf. A proportion of these dogs were also blind in one or both eyes since microphthalmia is another common side effect in M/M dogs. Although in many breeds it is possible to educate breeders to never breed two merle dogs together this advice is not possible to follow in Pomeranians since e/e dogs would not show the merle pattern. It would therefore be necessary instead to advise all persons who breed a merle dog to use only a black or sable mate or to have DNA testing done on their red, orange or white mate prior to breeding to be sure it did not carry merle."

Further complicating the picture is the case of breeding a merle to any dog with excessive white in the coat. Breeding merle to parti-color dogs with more than 50% white coat is advised against; especially if the merle parent also carries genes for particolor or "white spotting". It would be difficult to determine if the resulting puppies were merle or extreme piebald white-or both-without DNA testing! In either case, hearing can be affected.

Another factor to consider is that, according to a canine geneticist involved in a merle deafness study, the risk of deafness with merle increases when there are other coat color genes involved. Dogs that also have piebald are more likely to have problems.

DNA TEST FOR MERLE

We now have a DNA test for merle! This is a wonderful tool for breeders who choose to avail themselves of the technology. A cheek swab from the dog is sent to the lab, and they can tell you if your dog is merle or not. This test can also tell you if your dog carries one copy or two copies of the gene. Unfortunately, the US company offering this test closed its doors in March 2009. However, another company (Idexx) is poised to take over this service.

Unfortunately DNA testing remains costly, and may not be widely used even if available. AKC registration for Pomeranians now allows for designation of color as merle. However, merle status will continue to involve guesswork based on visual appearance until such time as the DNA testing becomes more widespread. Registration can not represent an accurate assessment of merle status, particularly in our breed, without this testing.

In the future, as the merle pattern becomes more popular and widespread in our breed, we may notice it will represent an ever-expanding portion of the gene pool.

HEARING TEST

The only way to know for sure if your dog is hearing impaired is through a Brainstem Auditory Evoked Response Test, or BAER test. This testing is only available at select facilities and can be expensive. It need only be performed once in a dog's lifetime (preferably at a young age) to rule out hereditary hearing impairment. A complete list of BAER testing facilities can befound on Dr. George Strain's website: www.lsu.edu/deafness/deaf.htm. Selective breeding for hearing dogs, as evidenced by normal BAER testing, can reduce the incidence of deafness in dogs, including those20with high risk factors.