By Kevin E. Noonan —

One of the benefits of the exponential increase in
genetic information consequent to the determination of the genomic DNA sequence
of numerous species (the various "genome projects" that include the
Human Genome Project) is that it permits sequence comparisons between related
species.  One result of this new
knowledge has been to verify the similarity of developmental control genes
throughout the animal kingdom (where genes like the Hox genes in nematode worms
and fruit flies have been identified in humans, for example).  Within species, decoded genetic
information, and the ability to associate genetic variation between individuals
in a species with specific phenotypes has elucidated the genes that control
such phenotypes.  Several examples
have been reported for domesticated dogs, where size is related to inheritance
of different alleles of the insulin-like growth factor-1 gene (IGF-1), and coat length, curliness, and
other features are related to inheritance of the FGR-5, KRT-71, and RPSO-2 genes.  Artificial human selection for breed characteristics has
contributed to the ability to productively perform these types of association
studies.

Leg length in dogs is another phenotypic feature
subject to the whims of human breed selection and characteristic of different
breeds.  In this week's edition of the journal Science, a group from the National Institutes of Health reports on
the genetic basis of this variation (termed chondrodysplasia), and associates
its results with a form of human dwarfish with similarities to canine chondrodysplasia
(wherein the growth plates in the long bones of the leg calcify prematurely,
producing shortened bones with a curved appearance) (Parker et al., 2009, "An Expressed Fgf4 Retrogene Is Associated with Breed-Defining Chondrodysplasia in Domestic Dogs," Science 325: 995-98).

The report is from Elaine Ostrander's lab at the
NIH, joined by researchers at the Department of Ecology and Evolutionary
Biology at UCLA, the WALTHAM Centre for Pet Nutrition in the UK, the Division
of Cardiovascular Medicine at the Oregon Health and Human Science University, the
Baxter Institute for Animal Health and the College of Veterinary Medicine and
the Department of Biological Statistics and Computational Biology, Cornell
University, and the Comparative Orthopedic Laboratory at the University of
Missouri.  The study is
particularly noteworthy as it identifies an expressed retrogene for fibroblast
growth factor 4 (FGF4); retrogenes, more
commonly termed pseudogenes, represent reverse-transcribed messenger RNA (thus
lacking introns) that have been re-introduced into the genome.  Until recently, these genes were
believed to be transcriptionally-silent relics of ancient retroviral infection
and were not expected to be transcribed.  It is now recognized that such retrotransposition can, and has occurred
at sites containing transcriptional regulatory sequences, as it appears to have
in these dogs, leading to a novel form of gene duplication.

Genome-wide association studies (GWAS) using
Affymetrix single nucleotide polymorphism (SNP) microarrays were performed on
DNA from 835 dogs comprising 76 distinct breeds.  Using American Kennel Club (AKC) breed standards, 95 dogs
from the chondrodysplastic breeds (including Pekinese, Welsh corgi, and
dachshund) were compared to a control group of 64 non-chondrodysplastic dog
breeds comprising 702 animals.  The
strongest association in these studies specific for the chondrodysplastic
breeds was found on canine chromosome 18 (CFA18).  This association was confirmed by studies on heterozygosity
in this region of canine chromosome 18, which was expected and was found to be
suppressed, consistent with human selection for chondrodysplasia as a part of
the breed standard for these breeds of dogs.  Upon further sequencing analysis, the region was found to
contain a processed retrogene of canine FGF4,
comprising all the coding exon sequences as well as the 3' untranslated region
(UTR) and a polyadenine tract (as expected from an mRNA retrotransposon), but
lacking the 5' regulatory sequences of the native canine FGF4 gene (which also resides on canine chromosome 18, about 30 Mb
from the FGF4 retrotransposon
insertion site).  This gene was
found in 175 dogs from 19 breeds exhibiting the chondrodysplasia phenotype, and
the gene was found to be homozygous in all but seven of these dogs.  Fortuitously, the group found a single
transition mutation difference (a G –> A change in the
retrogene) that permitted expression to be differentiated between the retrogene
and the native FGF4 gene.  Gene expression studies showed that the
retrogene was expressed in the articulate cartilage of the long bones from chondrodysplastic
dogs, "whereas only the G allele was detected in cDNA and genomic DNA
samples from non-chondrodysplastic dogs."

Finally, these authors found that the FGF4 retrogene was expressed using gene
regulatory sequences in a copy of a long interspersed nuclear element (LINE)
repeat residing at the retrogene insertion site.  However, analysis of the expression pattern of neighboring
genes in adult dog tissues showed that neither the native FGF4 nor the FGF4
retrogene were expressed in adult tissues.  Developmental regulation of FGF4 is believed to be mediated by sequences in the 3' UTR element
of the native gene, and the presence of these sequences in the FGF4 retrogene suggests that it is also
properly regulated during development.

All modern domestic dog breeds are believed to have
descended from the gray wolf, and the fact that chondrodysplastic breeds have
been developed several times independently (i.e., without direct ancestral
relationships) suggests that the retrotransposition event, and the presence of
the FGF4 retrogene, should be present
in populations of the ancestral gray wolf.  Using hapolotype analysis over the 24 kb region of
canine chromosome 18 comprising the FGF4
retrogene and its insertion site, "this combination was not found in any
of the non-chondrodysplastic dogs we tested but was identified in wolves from
Europe and the Middle East, supporting fossil evidence that these populations
contributed to the early development of the dog."

The authors put forth their hypothesis of how the FGF4 retrogene is involved in chondrodysplasia
in dogs, and its relevance to humans:

We hypothesize that
atypical expression of the FGF4
transcript in the chondrocytes causes inappropriate activation of one or more
of the fibroblast growth factor receptors such as FGFR3.  An activating
mutation in FGFR3 is responsible for
>95% of achondrodysplasia cases, the most common form of dwarfism in humans,
and 60 to 65% of hypochondrodysplasia cases, a human syndrome that is more
similar in appearance to breed-defining chondrodysplasia.  . . .  FGF4 induces the expression of sprouty
genes, which interfere with the ubiquitin-mediated degradation of the FGF
receptors including FGFR3, and
overexpression of the sprouty genes can cause chondrodysplastic phenotypes in
both mice and humans.

The greater significance of the study can be
summarized in no better words than the authors' own:

We have found a single
retrotransposition event producing a conserved, expressed retrogene that has
strongly focused the evolutionary direction of morphological change in the dog
because at least 12% of American breeds share a common phenotype and the
retrogene.  This retrogene is
actively segregating within the species, has a coding sequence that is
identical to that of the source gene, and to the best of our knowledge is the
only example of a functional retrogene found in morphologically distinct
populations of a single species that is actively maintained by selection.  If such rare mutational events or "sports,"
as Charles Darwin referred to them in "The Origin of Species," happen
only in the evolution of domestic animals, then these systems may be less
informative for understanding the origin of evolutionary novelty in wild
species.  However, if the molecular
phenomenon we have observed represents a class of genomic change associated
with dramatic phenotypic evolution, then such genetic changes might be keystone
molecular innovations.

    By Kevin E. Noonan —

The genetic age, characterized by numerous genome
projects (not the least of which is the Human Genome Project) has focused the
public's attention on the influence of our genes on our characteristics.  This has led to questions about whether
there is a "gene" for risk-taking, or homosexuality, or
aggressiveness.  Indeed, the
pervasiveness of the "gene" question for complex traits has produced
something of a backlash, illustrated by Richard Lewontin's books, Not in Our Genes and It Ain't Necessarily So, making the
reasonable point that genetic reductionism has its limitations.

But genes are certainly implicated in a number of
characteristics and traits, and one of the consequences of studying genomes has
been the elucidation of these relationships.  In 2007, a report in Science
disclosed that body size in domesticated dogs is associated with a particular
single nucleotide polymorphism (SNP) in the insulin-like growth factor I gene
(IGF-1) (see "From Toy Poodle to Rottweiler:  Why Is Fido So Small (or Large)?").  Last week, in Science, a group from the National Institutes of Health report on
another canine phenotype, coat variation, and the genes that are associated
with variations in several coat related traits (Cadieu et al., 2009, "Coat Variation in the Domestic Dog Is Governed by Variants in Three Genes," Science DOI: 10.1126/science.1177808).

The report, from Elaine Ostrander's lab at the NIH
and researchers at the Veterinary Genetics Laboratory at the University of
California, Davis, the Biology Department at the University of Utah, the
Department of Ecology and Evolutionary Biology at UCLA, Cornell University's
Department of Biological Statistics and Computational Biology, the Faculte de
Medicine in Rennes, France, and Affymetrix, involves genome-wide association studies (GWAS) for three coat
variations:  the presence or
absence of "furnishings" (eyebrows and mustaches as arise in wire-haired
dogs), coat length, and coat curliness.  The study involved more than 1,000 dogs from 80 breeds of domestic dogs,
and resulted in the identification of three genes associated with variations in
coat.  These genes are RSPO2, encoding R-spondin-2; FGF5, encoding fibroblast growth factor
5; and KRT71, encoding
keratin-71.  These genes, and
specifically SNPs associated with each of these genes, were found to be
associated with the presence or absence of furnishings (RSPO2), length (FGF5), and
curliness (KRT71).

Unlike the situation with body size, these three
genes form a combinatorial group of genes that can be independently segregated
(i.e., they all reside on different chromosomes) and can thus create a number
of different coat variations depending on the different variant alleles
inherited by particular breeds of dog (since coat characteristics are
frequently important components of the breed standard for different dog
breeds).  Three datasets were used
in these experiments:  the first
comprised DNA from 96 dachshunds varying as wire-haired (with furnishings),
smooth and long-haired coats; 76 Portuguese waterdogs, varying in curl type;
and 903 dogs form 80 breeds representing "a wide variety of [coat]
phenotypes."  The initial
results of polymorphisms segregating with the different coat traits were
validated using a larger panel of at least 661 dogs from 106 breeds that
included the appropriate controls.

From the dachshund (at right) studies was identified the
association of RSPO2 with furnishings
as a locus residing on canine chromosome 13 (CFA13).  As reported in the paper, "[a]
718 Kb homozygous haplotype in all dogs fixed with furnishings was located
within both the original 3.4Mb haplotype observed in the dachshund-only GWAS,
and a 2.8 Mb haplotype identified in crossover analysis within the dachshund
pedigree."  From this
chromosomal location the RSPO2 gene
was identified:

Fine-mapping allowed us to reduce the
homozygous region to 238 Kb spanning only the R-spondin-2 (RSPO2) gene,
excluding the 5'UTR and the first exon (Fig. 1D; fig. S1 and table S3).  RSPO2
is an excellent candidate for a hair growth phenotype as it is known to
synergize with Wnt to activate β-catenin
. . . , and Wnt signaling is required
for the establishment of the hair follicles . . . .  Moreover, the Wnt/β-catenin pathway
is involved in the development of hair follicle tumors or pilomatricomas . . . ,
which occur most frequently in breeds that have furnishings . . . .  Recent
studies have shown that a mutation in the EDAR gene, also involved in
the Wnt pathway, is responsible for a course East-Asian hair type found
in humans . . ., with some similarity to canine wirehair.

The polymorphism associated with the
furnishings trait in dachshunds and confirmed in 704 dogs of varying phenotype
is a 167 bp insertion within the 3' untranslated region (UTR) of the RSPO2 gene;
"297/298 dogs with furnishings were either homozygous (268) or
heterozygous (29) for the insertion, while all 406 dogs lacking the trait were
homozygous [for 3' UTR lacking the insertion]."  The authors also report a functional consequence of the
insertion, wherein there is a 3-fold increase in RSPO2 expression in muzzle skin from dogs with furnishings,
suggesting that the insertion in the 3' UTR influences mRNA stability.

For the association of FGF5 with hair length, the same type of
GWAS were performed, locating a polymorphism on canine chromosome 32 (CFA32) at
the FGF5 locus.  Here, the polymorphism affected the
coding sequence of the protein, resulting in a Cys –> Phe
change at a conserved residue (95) in exon 1 of the FGF5 gene (corresponding to a G –> T transversion mutation).  This association was not unexpected, as
prior work had shown that the FGF5
gene was associated with a long-haired or "fluffy" phenotype in Welsh
corgis.  Long hair was associated
with the TT genotype, consistent with a genetically recessive mode of
inheritance.  The T allele was
found in 91% of all long-haired dogs tested and in only 3.9% of short-haired
dogs (with medium-haired dogs having an intermediate 30% frequency level for
this allele).  Curiously, Afghan
hounds that have particularly long hair do not have the Cys95Phe mutation.

Finally, for the curly coat variation GWAS
were performed with Portuguese water dogs (PWD) (at left, First Dog Bo).  These studies revealed a SNP on canine chromosome 27 (CFA27)
located in the keratin 71 (KRT71)
genetic locus.  As reported in the
paper, "Non-curly haired dogs carried the CC genotype [and] curly coated
dogs had the TT phenotype," and all three genotypes (CC, CT and TT) were
found in PWDs, a breed that expressed curl hair to varying extents.  This SNP is also located in the coding
sequence (in the second exon) of the gene, causing an Arg –> Trp change
at residue 151.  These findings
were also rationalized by the authors:

Keratins are obvious
candidates for hair growth . . . and mutations in KRT71 have been
described in curly coated mice . . . .  The mutation described in our study is
within the second exon of the gene and may affect either or both of two protein
domains; a coiled-coil and a prefoldin domain.  Conceivably, sequence alterations in these domains could affect
cellular targeting, receptor binding or proper folding of the protein after
translation.

These three mutations were
sufficient to account for the coat phenotype in 95% of the dogs tested, a total
of 622 dogs representing 108 of the 160 dog breeds recognized by the American
Kennel Club.  The Supplemental
Materials contain a combinatorial diagram of the different variations at the
three loci that give rise to at least 7 different coat types (representing the
majority of coat types in modern breeds):

* The TT genotype was never found in this
combination, even though it would likely also display a short phenotype.

The authors set forth the following
conclusions.  First, the "ancestral
state" of all three genes (Cys95 in FGF5,
Arg151 in KRT71, and absence of an
insertion in the 3' UTR in RSPO2) is
found in short-haired dog breeds (as well as grey wolves, the ancestors of all
modern dog breeds).  Wire-haired
breeds (which all have furnishings) have the insertion in the RSPO2 gene.  Curly wire hair is found in dogs having both the RSPO2 insertion and the KRT71 mutation, while longer-haired dog
breeds have the FGF5 mutation.  Dogs carrying both the FGF5 mutation and the RSPO2 insertion have long, soft coats
and furnishings, whereas dogs with the FGF5
and KRT71 mutations have long,
curly coats.  Finally,
dogs bearing all three of the variants have long, curly coats, and furnishings.

Elucidation of this genetic architecture of
observable phenotypic traits is possible because of the large number of
different dog breeds and the great variability, on one hand, and the consistent
stability, on the other hand, of genetic transmission in these breeds.  This study suggests that these
analytical methods can (and will) be applied to assess the genetic bases of
other traits in dogs, and the extension of these results to homologous
phenotypes in other mammals including humans.

Photograph of mixed breed terrier (above) by Chris Barber, from the Wikipedia Commons under the Creative Commons license.