Leg Length Variation in Dogs and its Relevance to Human Mutations

    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.

Comments

[SKD]

Fascinating! Excellent post Kevin.

[Pacific Reporter]

What's up with all the dog posts? For a second I thought it was April 1st.

[Kevin E. Noonan]

Dear Pacific:

Well, I could say the blog is going to the dogs, but the reality is that I find the genetics to be fascinating. These studies are being done in dogs because they have so many specific breeds characterized by distinctive, stable, heritable phenotypes. But if we get similar studies on mice, cats or other animals, I will probably write about those as well.

I also think it's a nice break from FOBs, PTO rules packages, KSR and the rest of what we write about. These stories were posted on consecutive days just because I came across the journal articles at the same time. (We could have posted the dog fur story last Thursday, for example.)

Thanks for asking. We will now return to our regular programming.

[dog health]

Hi Kevin, the above study report was really helpful for me. I appreciate the efforts you take here.

- Dr. Mathew J.