**This webpage was produced as an assignment for an undergraduate course at Davidson College**Review of "Coat Variation in the Domestic Dog is Governed by Variants in Three Genes"
researchers set out to figure out what causes variations in fur
phenotypes in dogs using genomic approaches. They broke the
different variations into having or not having different combinations
of wire-hair/furnishings, long fur, and curly fur. Three sets of
dogs were used as the source of the data for the experiments: (i) 96
dachshunds which had dogs with wire-hair with furnishings (the mustache
and eyebrows some dogs have), smooth short hair, and smooth long
hair; (ii) 76 Portuguese water dogs that either did or did not have
curly fur; (iii) 903 dogs from 80 breeds with a wide array of
phenotypes (this data set was termed CanMap).
All three fur traits were mapped using the same general method:
A genome-wide association study (GWAS) was done within the breed data
set that displayed the trait being mapped to find the locus most
closely associated with the trait.
(2) A second genome-wide
association study was done with the CanMap data set to again find loci
closely associated with the trait in question.
within the closely associated regions further confined the area being
investigated as being specific to that trait.
(4) This constrained region was then sequenced to find specific mutations that could be the cause of the trait.
Results and Dicussion
Wire-hair & Furnishings
of the results of the two GWASs found a 718-kb area of variation
associated wire-hair and furnishings. The fine-mapping of this
region further cut down the common region to 238-kb, within which only
one gene was included: the R-spondin-2 (RSPO2) gene. RSPO2 is
a good candidate because it is involved in a pathway for the
establishment of hair follicles, and has also been tied to a specific
type of tumor most often found in dogs with furnishings.
Sequencing of RSPO2 from 7 breeds found a 167 base pair insertion in the 3' untranslated region (UTR) of RSPO2 in breeds with furnishings. The conclusion that the insertion mutation
is responsible for a dog having furnishings was supported by the fact
that 297 of the 298 dogs with furnishings were either homozygous or
heterozygous for the insertion and all 406 dogs without furnishings
were homozygous for the unmutated version of RSPO2.
This alignment also suggests a dominant mode of inheritance for
the trait, which would be in line with the proposed molecular effect of
the mutation: an increase in RSPO2
transcripts, an effect common for elements in the 3' UTR and supported
by analysis of tissue biopsies from furnished dogs. An increase
in transcripts would result in increased translation, and having more
than a normal level of protein is common in dominant disorders or
the GWAS of the the dachshunds data set and the GWAS of the CanMap data
set found regions of high association that included the FGF5
gene, which had been implicated in connection to hair length in
previous studies of dogs, cats, and mice. Fine-mapping of these
regions cut the region down to 67-kb, but still included the FGF5
gene. The single nucleotide polymorphism (SNP) with the highest
association, even after the region was sequenced, was in the first exon
and changed a
cysteine to a phenylalanine in the protein product of the gene.
The mode of inheritance for the the trait appears to recessive as
all long-haired dogs were homozygous for the SNP, while short or
wire-haired dogs could be either heterozygous or homozygous for not
having the SNP. This mutation is the only cause of long hair in
dogs, as 3 breeds with long hair did not have the mutation or show any
association to the area near the gene.
a GWAS within the Portuguese water dog population and a GWAS within the
CanMap found SNPs close to each other on chromosome 27.
Fine-mapping of the region between the two SNPs found that the
area included two keratin genes. Sequencing of most of the region
found a specific SNP in the KRT71
keratin gene that was very closely associated with the trait. The
SNP occurs in the protein-encoding part of the gene and changes an
arginine to a tryptophan. This alteration could affect cellular
targeting, receptor binding, or proper folding of the protein.
The conclusion that this mutation is a cause of curly hair is
supported by previous experiements, which have described mutations in KRT71 in curly haired mice.
combinations of these three mutations were found to account for the fur
phenotypes of 95% of the dogs tested, which included 108 breeds out of
the approximately 160 recognized by the American Kennel Club.
They typed 622 dogs for the 3 mutations they found, and the
mutations explained the phenotype for each dog:
None of the 3 mutations: short-haired breeds
RSPO2 mutation only: wire-hair and furnishings
RSPO2 and KRT71 mutations: curled or kinked wire-hair
mutations: long soft coats with furnishings
FGF5 mutation only: long, straight hair
FGF5 and KRT71 mutations: long and curly coats
All 3 mutations: long and curly with furnishings
mutation was not found by itself in any curly haired dogs. The
authors' reasoning behind this was that the hair had to be of a certain
length to curl. None of the three mutations isolated in the
experiments were found when gray wolves were typed for them, suggesting
that they are not the ancestral versions of the genes. The high
degree of similarity in the areas around the mutations suggests that
each mutation occured once and then was transferred to other breeds
recently, likely due to artificial selection by humans.
1A- This is just a pictorial representation of dachshunds exhibited the
fur phenotypes that the authors used the population to investigate:
smooth-coat on the left, long hair in the middle, and wire-hair with
furnishings on the right. This figure just serves as a
visualization for the reader of what the authors are referring to when
discussing long, short, and wire-hair.
Figure 1B- This is a
graphical representation of the GWAS performed within the dachshunds
population in order to find regions associated with wire-hair.
The x-axis is the genome, with each gap between hashes being one
chromosome, and the y-axis is the negative log of the P value.
The P value is the chance that the variation and the trait are
associated purely by chance, so the authors were looking for what
locations had the smallest P values, the smallest chance that any
association observed was simply due to chance. The negative log
of a small value is large, so the highest peaks are places of the
lowest chance that the observed connection between variation and trait
is due to random chance.
Figure 1C- This figure is similar to
1B, the only difference is that the data set used to generate this GWAS
was the CanMap data set. The y-axis scale is also 10x the scale
on the 1B, showing that this association is more strongly coorelated
with wire-hair, likely an effect of having almost 10x as many dogs in
the CanMap data set as in the dachshunds data set.
This is a representation of each step they took to limit down the
section that needed to be sequenced, and also what genes are found in
the shared regions. The red rectangle is the associated region
found with the dachshunds data set, the blue rectangle is that could
with the CanMap data set, and the green rectangle is the result of
fine-mapping between the two to find an area of homozygosity between
the two. Below the green rectangle the insertion found through
sequencing and its location is shown. The open boxes on the
bottom of the figure show genes that are in the regions covered by any
of the rectangles. We can see that by the time the region is
limited all the way down after fine-mapping, only the RSPO2 gene is within the region.
2A- The layout of this figure resembles that of 1D because it is
showing the same things, just for hair length instead of wire-hair.
The color coding for rectangles is the same as 1D: red is from
dachshunds GWAS, blue is from CanMap GWAS, and the green is the result
of fine-mapping between the two. The most strongly associated SNP
is shown in it's position within the region and gene, and the open
boxes again show genes in the regions found to be related to the trait
at various steps along the method.
Figure 2B- These are more
pictures to give the reader an idea of exactly what fur trait, curly
hair, is being investigated by the authors.
Figure 2C- This is
supposed to be showing the same idea as 1D and 2A except for curly
hair, but is cut down from those as it only shows the green rectangle
of commonly shared area found after fine-mapping, the strongest
associated SNP after fine-mapping, the strongest SNP association after
sequencing, and the genes within the green rectangle. It shows
that both stron SNPs found are within the KRT71 gene.
figure is the summary figure for the paper. The table shows all
the combinations of mutations found by the authors, and resultant
phenotype labeled with a letter that cooresponds to a picture of a dog
exhibiting that phenotype.
paper appeared to do a good job showing that the mutations they
isolated are the causes of the traits they investigated. I would
be very hesitant to say that these 3 mutations are the cause of all the
fur phenotypic diversity in dogs, as they acknowledge that some dogs'
phenotypes were not explained (specifically some long-haired dogs), and
they only typed about 2/3 of the AKC breeds, which do not include other
unrecognized breeds. I also wish that the GWAS data was included
in the figures for each trait, instead of only for wire-hair.
Though the authors make no attempts no extrapolate beyond dog
hair phenotypes, some people might try to use the paper to say
something about humans, which would be risky at best because a lot of
the ability for a single mutation to be associated with a trait stems
from the high level of past artificial selection in dogs, a feature
that has not occured in humans. Overall this was a good paper
that I found very interesting.
Cadieu E, Neff MW, Quignon P, Walsh K, Chase K, Parker HG, VonHoldt BM, Rhue A, Boyko A, Byers A, et. al. Coat variation in the domestic dog is governed by variants in three genes. Science. 2009; 236:150-153.