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An Expressed Fgf4 Retrogene Is Associated with Breed-Defining Chondrodysplasia in Domestic Dogs

A summary of the original research article:

Parker, Heidi G., Bridgett M. VonHoldt, Pascale Quignon, Elliott H. Margulies, Stephanie Shao, Dana S. Mosher, Tyrone C. Spady, et al.. 2009. “An Expressed Fgf4 Retrogene Is Associated with Breed-defining Chondrodysplasia in Domestic Dogs”. Science 325 (5943). American Association for the Advancement of Science: 995–98. http://www.jstor.org/stable/20544365.


Introduction

During the evolution of a genome, the retrotransposition of processed mRNAs is a common source of acquiring new genes. Although a majority of these new genes lead to debilitating mutations, there is a small percentage that can encode functional proteins. In domestic dogs with short legs, also known as chondrodysplasia, it was found that a retrogene that encoded fgf4 was present. The experiment that was done to come up with these results was a multi-breed association analysis. A total of 835 dogs from 76 distinct breeds provided maximal coverage of phenotypic variation and were genotyped by using the Affymetrix version 2.0 SNP chip. The cases that consisted of chondrodysplastic breeds comprised of 95 dogs from eight breeds while the control, or non-chondrodysplastic group included 702 dogs from 64 breeds. A single-marker analysis showed a strong association between a SNP on chromsome 18 and the short-legged phenotype. The researchers also compared heterozygosity in 139 chrondrodysplastic cases and 173 controls and found that those that were chondrodysplastic had lower amounts of heterozygosity than then controls.

Shows the comparison of wildtype dogs with dogs possessing the chondrodysplastic phenotype

Experiment and Results

Researchers sequenced 54 amplicons in 44 dogs from 20 breeds, with 18 being control subjects and 26 being test subjects (dogs which exhibit chondrodysplasia.) The focus of the sequencing was to identify additional SNPs, causative mutations, and locating the smallest, common haplotype amongst tests subjects. The team used numerous methods to test for and confirm the expression of the Fgf4 retrogene. Techniques included the usage of pcr reactions, Illumina Genome Analyzer, Velvet algorithms, Sanger sequencing, as well as BLAT analysis. The results identified 123 SNPs in the test subjects, of which 50 formed one continuous homozygous haplotype. This identified region accounted for approximately 24 kb. Within the haplotype scientists discovered a portion of the 3’UTR, a pseudogene, and two evolutionary conserved sequences. A 5kb insert was found to be shared amongst 19 of the 20 breeds and the scientists propose that it is decidedly associated with chondrodysplasia. The finding also noted, “a single contig with complete alignment at 100% identity to Fgf4, which is located on CFA18…about 30 Mb form the insert.” The Sanger sequencing confirmed that the identified insert contained a retrogene but did not contain the expected introns or promoter sequence; it did however included the unmodified exons, the 3’UTR, and poly A tail. Analysis of retrogene-specific cDNA at “a position syntenic to chr18:5144160, 455 bp distal to the coding sequence” noted that the source gene contained a G nucleotide at that position but the retrogene contained an A nucleotide. The scientist discovered that chondrodysplastic dogs contained both A and G alleles in the generated cDNA, but non-chondrodysplastic dogs only contained a G nucleotide.

A dog possessing the Fgf4 gene that encodes chondrodysplasia.
Chart shows the comparison between chondrodysplastic dogs (red) and non-chondrodysplastic dogs (black) and its associated region on chromosome 18.

Analysis

After sequencing the common haplotype among short-legged dogs, it was found to be a retrogene to the Fgf4 gene. This special sequence was expressed in the cDNA of the dog breeds with chondrodysplatic and was absent in those breeds with proportional legs. Additional research found that the Fgf4 retrogene and Fgf4 gene are only expressed in the cartilage of fetal and neonatal dogs, the expression is time-sensitive and is not found once the dog has reached adulthood. In humans and in mice, dwarfism similar to those seen in these dog breeds is due to a mutation of the FgfR3 growth factor receptor, this degradation are caused by an overexpression of the "sprouty genes" as a result of the Fgf4 gene.

Chromosome map showing the location of the Fgf4 gene - likely to be positioned on chromosome 18.

Conclusions

The vast number of dog breeds in today's society were created in countries all over the world for a wide variety of reasons. While the short-legged breeds do not necessarily stem from the same original ancestor, they all share the haplotype of the Fgf4 retrogene near the FGF4 source gene. This is a unique discovery, in that it is one of the only examples of a specific retrogene being largely responsible for a big physical discrepancy among members of the same species.

Source

1. Parker, Heidi G., Bridgett M. VonHoldt, Pascale Quignon, Elliott H. Margulies, Stephanie Shao, Dana S. Mosher, Tyrone C. Spady, et al.. 2009. “An Expressed Fgf4 Retrogene Is Associated with Breed-defining Chondrodysplasia in Domestic Dogs”. Science 325 (5943). American Association for the Advancement of Science: 995–98. http://www.jstor.org/stable/20544365.