Basic Genetics III: Linkage and Crossing
Over
Up until now we have assumed that all genes were inherited
independently. 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
independent 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 will 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 independently, 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 maa being the gene
associated with the healthy gene, and mab being the marker
that seems to be associated with the defective gene, both being true for
the test population. For the genes actually producing the problem, we
will use H, with Hh being the normal, healthy gene and hd
being the recessive gene which causes the problem. In the original test
population, maa was always on the same chromosome with Hh,
and mab was on the same chromosome with hd. In
other words, chromosomes are either maaHh or mabhd,
never maahd or mabHh. If a
dog has maa on both chromosomes, it is also Hh on
both chromosomes, a genetic clear. If it has maa on one
chromosome and mab on the other, it also has one Hh
gene and one hd gene, and is a carrier. If it has mab
on both chromosomes, it has hd 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 maaHh or mabhd
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 maahd
and/or mabHh. 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, (mabHh) 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 (maahd) 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 origin) 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.
