Basic Genetics III: Linkage and Crossing Over

Up until now we have assumed that all genes were inherited independantly. However, we have also said that genes are arranged on chromosomes, which are essentially long strands of DNA residing in the nucleus of the cell. This certainly opens the possibility that two otherwise unrelated genes could reside on the same chromosome. Does independant inheritance hold for these genes?

To start with, we need to consider the rather complex process that forms gametes (egg and sperm cells, each with only one copy of each chromosome) from normal cells with two copies of each chromosome., one derived from each parent. I am not going to go into the details, beyond remarking that at one stage of this process, the maternally-derived chromosome lines up with the corresponding paternally-derived chromosome, and only one of the two goes to a specific gamete. If this were all there were to it dogs, having 39 chromosome pairs, would have only 39 "genes", each of which would code for a wide variety of traits. In fact, things are a little more complicated yet, because while the paternal and maternal chromosomes are lined up, they can and do exchange segments, so that at the time they actually separate, each of the two chromosomes will most likely contain material from both parents.

At this point we need to define a couple of terms. Two genes are linked if they are close together on the same chromosome and thus tend to be inherited together. Linkage in common usage, however, may apply to a single gene having more than one effect. An example which is not linkage in the sense used here is the association between deafness and extreme white spotting. White spotting is due to the melanocytes, the cells which produce pigment, not managing to migrate to all parts of the fetus. Now it turns out that in order for the inner ear to develop properly, it must have melanocytes. If the gene producing white spotting also prevents the precursors of the melanocytes from reaching the inner ear, the result willl be deafness in that ear. In other words, the same gene could easily influence both processes. Thus deafness and white spotting are associated, but they are not linked. They are due to what is called pleiotropic (affecting the whole body) effects of a single gene.

In true linkage, there is always the possibility that linked genes can cross over. Imagine each chromosome as a piece of rope, with the genes marked by colored stripes. The matching of the maternal and paternal chromosomes is more or less controlled by the colored stripes, which tend to line up. But the chromosomes are flexible. They bend and twist around each other. They are also self healing, and when both the maternal and paternal chromosomes break, they may heal onto the paired chromosome. This happens often enough that genes far apart on long chromosomes appear to be inherited independantly, but if genes are close together, a break is much less likely to form between them than at some other part of the paired chromosomes.

Such breaks, called "crossing over" do occur, and occur often enough that they are used to map where genes genes are located on specific chromosomes. In general, neither linkage nor crossing over is of much importance to the average dog breeder, though one should certainly keep in mind the possibility that the spread of an undesirable gene through a breed is due to the undesirable gene being linked to a gene valued in the breed ring. Crossing over is also important in the use of marker genes for testing whether a dog carries a specific gene, most often a gene producing a health problem.

There are two distinct ways of using DNA testing to identify dogs carrying specific, undesirable genes. The first (and preferable) is actually to sequence the undesirable gene and its normal allele. This allows determination of whether the dog is homozygous normal, a heterozygous carrier, or homozygous affected. Since the genes themselves are being looked at, the results should be unambiguous. (The breeding decisions based on these results are still going to depend on the priorities of the breeders.)

In some tests, however, a marker gene is found that appears to be associated with the trait of interest, but is not actually the gene producing that trait. Such a marker is tightly linked to the gene actually causing that trait. This does not work at all badly providing that the group on which the test was validated is closely related to the group to which the test was applied. Use of this type of test on humans usually requires that the test be validated on close relatives, and applied only to people closely related to the validation group.

It is true that dogs of a given breed tend to be closely related to each other. However, the breed-wide relationship is generally through more distant ancestors than most people can trace in their own genealogy. In Shetland Sheepdogs, for instance, almost all US show stock can be traced to dogs imported from the British Isles between 1929 and 1936, with only a tiny influence of imports after 1950. This means that a crossover appearing on one side of the Atlantic since 1950 (20 or so dog generations) might not show up on the other side. Marker tests that work on U.S. populations might not work at all on British dogs, or on a dog with recent British ancestry.

Even without physical separation here is always the possibility that at some point in the breed history a crossover occurred. Quite a large fraction of the breed may have the original relationship between the marker gene and the problem gene, but if a crossover occurred in an individual who later had a considerable influence on the breed, the breed may also contain individuals in which the marker gene is associated with the opposite form of the problem gene. Since the relationship between individuals of the same breed may go back 30 generations or more, and there is a chance of a crossover occurring in each generation, linked markers need to be used with caution and with constant checking that marker test results correlate with clinical results.

Let's look more closely at this.

Let our marker gene be ma, with ma^a being the gene associated with the healthy gene, and ma^b being the marker that seems to be associated with the defective gene, both being true for the test population. For the genes actally producing the problem, we will use H, with H^h being the normal, healthy gene and h^d being the recessive gene which causes the problem. In the original test population, ma^a was always on the same chromosome with H^h, and ma^b was on the same chromosome with h^d. In other words, chromosomes are either ma^aH^h or ma^bh^d, never ma^ah^d or ma^bH^h. If a dog has ma^a on both chromosomes, it is also H^h on both chromosomes, a genetic clear. If it has ma^a on one chromosome and ma^b on the other, it also has one H^h gene and one h^d gene, and is a carrier. If it has ma^b on both chromosomes, it has h^d on both chromosomes and is a genetic affected. At least, that is the assumption on which marker tests are based.

Now suppose that at some point a crossover occurred between the ma and H loci. The probability of a crossover may be very small in any individual breeding, but remember that there are a lot of breedings behind any particular dog. We can still assume that most of the chromosomes will still be of the ma^aH^h or ma^bh^d type, or the original validation of the marker test would have failed. But now suppose that a small fraction of the chromosomes are of types ma^ah^d and/or ma^bH^h. We now have four chromosome types, and sixteen possible combinations. Some of these will test the same, since the only difference is in which chromosome comes from the mother and which from the father, but there are still sixteen possible outcomes. In the table below both the marker results (upper) and the true results (lower) are shown for each possible combination:

	maaHh	mabhd	maahd	mabHh
maaHh	clear maamaa	carrier maamab	clear maamaa	carrier maamab
	clear HhHh	carrier Hhhd	carrier Hhhd	clear HhHh
mabhd	carrier maamab	affected mabmab	carrier maamab	affected mabmab
	carrier Hhhd	affected hdhd	affected hdhd	carrier Hhhd
maahd	clear maamaa	carrier maamab	clear maamaa	carrier maamab
	carrier Hhhd	affected hdhd	affected hdhd	carrier Hhhd
mabHh	carrier maamab	affected mabmab	carrier maamab	affected mabmab
	clear HhHh	carrier Hhhd	carrier Hhhd	clear HhHh

Note that in only six of the sixteen possible types is the marker indication of genotype correct. If the crossover genotypes are rare (as would normally be the case if the marker test verified at all) most of the population will be in the upper left quarter of the table, where the marker will correctly predict the true genotype. But if any of the chromosomes trace back to a crossover, a marker test may give a false sense of security (carrier or affected shows clear by marker testing) or result in discarding a healthy dog (carrier or clear shows affected or carrier by marker testing.)

If only three chromosome types are available, the two verifying types plus one crossover, then if the marker gene is associated at times with the healthy allele, (ma^bH^h) the result will include dogs which are affected or carriers by marker analysis which are genetically carriers or clears (false positives.) If the other chromosome type has the undesirable allele not always associated with the marker (ma^ah^d) the results will include dogs clear or carriers by marker analysis that are actually carriers or affected (false negatives.) However, the existance of one crossover chromosome type would make me suspicious that the other might also exist in the breed.

So are marker tests of any use at all?

Yes! In the first place, they demonstrate that the actual gene is on a relatively limited portion of a known chromosome. The marker gene can thus assist in finding and sequencing the gene actually causing the health problem.

In the second place, marker tests are accurate so long as neither parent of an individual has a crossover chromosome. In humans, such tests are most likely to be used when a problem runs in a particular family. The linkage of a marker with the genes actually producing the problem is generally based on studies of how the marker is linked to the genes in that particular family. With dogs, the verification is normally done on a breed basis, and the fact that breeds may actually be split into groups (color, size, country of origen) which interbreed rarely if ever is likely to be ignored. Dogs closely related via close common ancestors to the test population are the best candidates for marker testing. In general, keep up conventional testing side by side with the marker testing. If the marker testing and the conventional testing disagree (e.g, affected dog tests clear or clear dog tests affected) consider the possibility of a crossover, and notify the organization doing the test.

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