Archive | January, 2014

Classical versus molecular genetics

Molecular

As an artist, I always found variations in animal coloration fascinating, but I fell in love with coat color genetics in 1989 when I purchased a copy of Horse Color (1983) by Dr. Phillip Sponenberg and Bonnie Beaver. The book contained what seemed at the time to be countless color variations and a structure for categorizing them. I read the book cover to cover, and then set out to find as many of the referenced books and papers that appeared in the bibliography in the back. The whole system seemed so logical, and the basic framework fairly easy to understand.

HorseColor183
One of the pages of color plates from my very worn copy of Horse Color. It is a good thing that eyeballs do not absorb printing ink, or this page would have ended up blank years ago!

Because it is in my nature to share information, it was not long before I was writing about horse color with the intention of teaching others that same framework. And that was how I usually presented the subject: “This is pretty simple.” I could explain the concept of base colors–black, chestnut and bay/brown–and then the modifiers that altered those colors, either by diluting the color itself, or adding white hairs, or covering them with a white pattern. All of those were governed by the rules of genetic inheritance most of us learned in high school biology, when we used Punnett squares to map out the ratios of green and yellow pea pods.  Those ratios would tell us if something was dominant or recessive, or incompletely dominant, or perhaps a homozygous lethal. All that was needed was to tie that knowledge to an eye for the nuances of shade and pattern, and you were set!

Using this approach, I could give an hour-long presentation without using scientific terms like allele or epistasis. Instead I used a system that relied on visual understanding, because I found that many non-scientists would shut down if the initial information was abstract or overly technical. Most of the questions I encountered had pretty straight-forward answers, so this tended to work well. It was easy enough to expand on the basic concepts with technical information once someone was more comfortable with the subject. But somewhere along the way, explaining horse color became a lot more complicated. Over time, I found that even the most basic questions required increasingly more technical answers.

OldSlides2
Cycling through the same set of images, each showing the effect of a given modifier on each of the base colors, was one of the most effective ways to communicate the concept of modifiers. (It could be argued that my use of Comic Sans was perhaps less effective.)

So what changed? Why has color genetics become so complex and so much more technical?

The answer is that there has been a change in the field of genetics. Mendelian genetics, which is also known as classical genetics, predates the advent of molecular biology. The huge leaps in our understanding of coat color in mammals–horses included–come from advances in molecular biology. When horse color research relied on classical genetics, it was pretty easy to explain the subject in simple terms; after all, the original discoveries were made before even the most basic concepts about sexual reproduction were understood. But the science has advanced far past that point, making it not only possible to find the exact, physical mutation in the genetic code, but to understand why that particular change caused the final result that we can see. 

The albino Dobermans are a good example of this difference. Classical genetics could tell breeders that the trait was recessive, and the visual appearance of the dogs suggested that the dogs carried some form of albinism. Molecular genetics makes it possible to tell what kind of albinism (or dilution, if you prefer) is involved. That makes it possible to test for carriers, but it also allows comparisons with other dilutions to help determine what, if any, detrimental effects might occur because of the mutation. As more is known about the function of the different genes–and each mutation found adds to that body of understanding–it becomes easier to predict what might be directly caused by the change, what might be linked, and what might be unrelated. In horses, this kind of research determined that Multiple Congenital Ocular Anomalies (MCOA), formerly known as Anterior Segment Dysgenesis (ASD), was “tightly linked” to the silver dilution, and that homozygous silvers were at greater risk for eye defects regardless of their breed.

Another good contrast between classical genetics and molecular genetics is the prediction of lethality. In a comment from the last post, a reader asked about the belief that some forms of Dominant White were lethal in their homozygous form. In classical genetics, crosses that produced early lethals (that is, lost pregnancies rather than offspring that did not survive long) were determined by the absence of true-breeding individuals, and a ratio that indicated that one portion of the expected outcome was missing (2:1 instead of 3:1). While those factors are still considered, knowing what role each gene plays in the development of the organism allows researchers to predict outcomes in a way that just looking at production ratios cannot. If the gene is responsible for task A, B and C during development, and if shutting its function down at point X ends that process before reaching C, and if C is necessary for life, then it can be assumed that without a normal copy of the same gene (ie., if the animal is homozygous for that mutation) the resulting offspring is non-viable. That is why some recent studies have suggested that certain crosses might not be viable, even when the mutations themselves are rare enough that there are not statistically significant numbers to assess ratios, and where a lack of true-breeding animals might not be particularly informative.

Lethal
This Punnet Square of Lethal Yellow in mice illustrates how homozygous lethals change the expected ratio from 3:1 to 2:1. Instead of the three yellow (two heterozygous and one homozygous) and one white, the result is two yellow to one white. 

For breeders, this level of understanding holds a lot of promise for the future, because it makes it possible to analyze the connection–or lack of connection–between a color and undesirable traits. In the past, defects have been tied to colors, often using nothing more than rumor or supposition. It was not unusual to see a superficial similarity, like white patches or lighter eyes, used to suggest that a given color was susceptible to the same defects. This is how a diluted dog becomes an “albino” carrying something that is “a defect in all species”. Molecular genetics offers the opportunity to examine the actual cause for the reduction in pigment, and a means of determining just exactly what other problems, if any, this alteration might create. Yes, this does mean that more often the answer to questions about color will be, “It is complicated,” rather than “It really is pretty simple.” But knowing the true nature of a given mutation, and being able to identify it, gives breeders more control when it comes to obtaining, or avoiding, certain colors. And instead of culling all suspect animals, and losing whatever else they might have to contribute to their breed, breeders can make more informed selections whatever their goals are in terms of color. Seen in that light, the increased complexity of modern genetics seems a small price to pay.

(Punnet Square graphics courtesy of Wikimedia Commons, with apologies to my rodent-loving readers. Molecular graphic courtesy of the U.S. National Library of Medicine.)

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Is this still a horse color blog?

Talking

So what do albino dogs have to do with horse color? Certainly the fact that there are “cremello” Dobermans is interesting, but why is that relevant to this blog?

The story about Angel and her unusual coloring brought together a couple of thoughts that have been on my mind lately. I thought I might explain them briefly here, and then expand on each in a separate post.

The shift from Mendelian Genetics to Molecular Genetics
When Pearson and Usher studied albinos, Mendel’s work had been only recently rediscovered. The results from their experiments with the Pekingese left them convinced that the problem was too complex for Mendel’s theories to accurately predict the outcome of their crosses. There were too many variables. In the hundred years since, science has not only identified many of the variables that eluded the two men, but it has begun to unlock the underlying mechanisms that produce the colors. Beyond just making modern genetic tests possible, this focus on the molecular level means that it is possible to understand the nature of color mutations in ways not possible before. This has implications for anyone interested in sound and ethical breeding decisions.

Just how many colors are there? 
It is clear when reading A Monograph on Albinism in Man and Albinism in Dogs that the authors were not only grouping together different dilutions in dogs, but also blue-eyed white dogs that were in all likelihood spotted or merled. Because it is possible to test for specific colors, we now know that some colors that look alike are different things, at least on a genetic level. In some cases colors have proven to be unrelated even though the end result can be difficult to distinguish. Still other things are genetically related, in the sense that they are mutations to the same gene, but they differ visually – sometimes dramatically so. In other cases, they may be visually similar, but their pattern of inheritance is quite different. As a result, the list of known (and suspected) colors and patterns has been growing rapidly, while grouping and categorizing them has become more challenging.

What do we call all these new colors?
With each new discovery, the old naming system has been strained. Just as Dilution is a color in dogs, but a category of (visually) related colors in horses, many of the colors  - particularly the pinto patterns – could more accurately be termed categories now. Yet many still speak of patterns as singular things. The most obvious problem has been with the what was once called sabino, and now sometimes jokingly referred to as KMOSS (“KIT Mutation of Some Sort”). What do we call these new categories, and the colors within them? This last topic should make an appropriate segue into the new paper on white markings, since that, too, is part of the same problem.

So bear with me for a few days while I pull these related thoughts together in a (hopefully) coherent way, and in no time we will be back to talking about horses again!

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Cornaz albinos

Patty
Patty, an albino Pekingese from an early 20th century experimental breeding program

So the mutation responsible for white Dobermans has been identified, and is similar to the Cream and Pearl dilutions in horses. So what about Angel, the albino Shih Tzu that started this discussion? Is she also the canine equivalent of a cremello?

Probably not. An albino Lhasa Apso tested negative for the mutation found in Dobermans. What’s more, she did not have a mutation to that gene (SLC45a2). Whatever caused her pink skin and cream coat, it appears to be unrelated to the color in Dobermans. Because the Shih Tzu and Lhasa Apso shared a stud book up until 1934, it seems more likely that Angel would have the same mutation as the Lhasa. Both breeds also share a history with the Pekingese, which has one of the most throughly documented families of albino dogs. The influence of that family may explain why albinism in dogs is often seen in the smaller Asian breeds.

The albino Pekingese were the focus of an experimental breeding program conducted around the turn of the last century. Extensive information on the foundation animals appears in A Monograph on Albinism in Man, published in 1913. Although the program was disrupted by World War I, it did continue for a time and a follow-up article was published in 1929. Because the information was so detailed, it is possible to know the founder for the color in the Pekingese.

AhCum

That is Ah Cum, the “grandfather” of the Pekingese breed. He was an ordinary red sable, but because he was the common ancestor in all the known albinos in that breed, the authors of the study believed that the albino gene came from him. There can be little doubt that his son, Ch. Goodwood Lo, carried the recessive gene for the color.

Lo

What is interesting about this particular family, and this experiment, is that many of the dogs were photographed. The written notes on the dogs can be less than helpful, because those studying the dogs did not yet understand something that those of us who study animal coloration take for granted now, which is the concept of base colors and modifiers. So instead of seeing these dogs as a basic color, like sable or black-and-tan, that had been diluted down to a nearly white color by a modifying gene, the researchers assumed they were dealing with separate colors. They called the near-white dogs “Dondo Albinos” and the somewhat darker dogs “Cornaz Albinos”. That latter term is still used for this color in many breeds where albinos are known to occur.

It should be noted that the authors knew these dogs did not have pink eyes, or even necessarily blue ones. They considered an eye to be albinotic if the pigment was reduced. In fact, their discussion of equine eyes touched on a question that has often been on my mind. We often hear that horses do not have “true albinism” because there has not yet been a documented case of pink eyes. What I have often wondered was whether a pink eye is actually possible in all animals given the varying structure of the eye. Is an eye without pigment always pink or red?

In regard to the colour of the iris as seen during life in the imperfectly albinotic eyes, the present observations confirm in an interesting manner our previous knowledge that when the mesoblastic pigment is absent the iris is either white (the so-called “wall” eye) or blue or slaty blue according to its thickness and texture, a thick and fibrous iris being white and opaque throughout or translucent only at its thinnest part. In the horse even the thinnest or pupillary zone is probably too thick to be translucent.

Here the authors – two of whom are ophthalmologists – seem to suggest that in some animals an eye without pigment might not necessarily appear pink or red. Yet they also mention the difficulty in finding a horse with perfectly unpigmented eyes.

We have hitherto not succeeded in meeting with a perfect albino horse; the epiblastic pigment of the iris seems peculiarly persistent.

The Pekingese family was considered an example of ‘imperfect’ albinism, which meant that there was some trace of pigment either in the eyes, skin or hair. That is still what this kind of coloring is called in the dog world: albino. If something like this turned up in the horse world, there is little doubt that it would be considered a dilution, just as champagne and pearl were when they were identified. But as I mentioned, when this breeding program was undertaken the concept of a diluting modifier was not understood. (To give some perspective on the understanding of inheritance at the time, James Cossar Ewart’s famed Penycuik Experiments disproving telegony – the idea that previous matings left a taint that could influence later offspring – had been published only a dozen years earlier. Crick and Watson’s discovery of the double-helix structure of DNA was still forty years away.)

To a modern student familiar with how diluting modifiers work, the underlying colors on some of these Pekingese is obvious. Hints of the dark ‘spectacle’ markings common in sable Pekingese can be seen in the photo of the dog at the top of this post. The dog below looks to be a dilution of the black-and-tan pattern, judging from the coloring on the face and forelegs.

Fo

It also appears that some of the darker dogs may have been carrying some combination of the Cornaz albino dilution and the more common dog dilution, Brown. The color of the darker Cornaz albinos was described as “scraped chocolate”. One of the ancestors of the foundation stock was described as “liver and white”, and there was at least one puppy from the experiment that was noted as having a brown, not pink, nose. When later generations were crossed on black Pomeranians – which the researchers, anticipating the “designer dog” trend by a hundred years, called Pompeks – one of the first generation litters resulted in two chocolate puppies. In this way, it seems possible that the Cornaz dilution combines with Brown to produce an intermediate shade, much like Cream combines with Pearl or Champagne in horses.

Some of the puppies were surprisingly dark at birth, but still had pink – not chocolate – noses. The authors noted that the color at birth tended to be darker than the mature color, which is also true for Champagne foals. This Japanese Chin shows the kind of deeper coloring that some of the adult dogs in the study were said to have. Although it is not (yet) possible to test for the Cornaz coloring, it would be interesting to test some of the darker Cornaz albinos for Brown.

Fe

One thing that I have found surprising, since the initial post about Angel, is the number of albino-like dogs, and the range of breeds where they have occurred. It is possible that some do share the same mutation as the Dobermans, either due to outcrossing or because the mutation predates the formation of those breeds. Others likely share whatever mutation is responsible for the albino Lhasa Apso. It is also possible that there are still more mutations unrelated to the one in Dobermans and the one in the Asian breeds. With the exception of Pearl, dilutions in horses have so far proven to be dominant, or at least incompletely dominant. Because the diluted colors in dogs are more often recessive, it is far easier for them to hide for generations, especially when they are rare in the population. If these are older dilutions, then it is possible that albinos may appear unexpectedly in different breeds, just as chocolates and blues do.

So what does this all have to do with horses? That’s the topic for the next post.

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