Multiple mutant T alleles cause haploinsufficiency of Brachyury and short tails in Manx cats

Abstract

Most mammals possess a tail, humans and the Great Apes being notable exceptions. One approach to understanding the mechanisms and evolutionary forces influencing development of a tail is to identify the genetic factors that influence extreme tail length variation within a species. In mice, the Tailless locus has proven to be complex, with evidence of multiple different genes and mutations with pleiotropic effects on tail length, fertility, embryogenesis, male transmission ratio, and meiotic recombination. Five cat breeds have abnormal tail length phenotypes: the American Bobtail, the Manx, the Pixie-Bob, the Kurilian Bobtail, and the Japanese Bobtail. We sequenced the T gene in several independent lineages of Manx cats from both the US and the Isle of Man and identified three 1-bp deletions and one duplication/deletion, each predicted to cause a frameshift that leads to premature termination and truncation of the carboxy terminal end of the Brachyury protein. Ninety-five percent of Manx cats with short-tail phenotypes were heterozygous for T mutations, mutant alleles appeared to be largely lineage-specific, and a maximum LOD score of 6.21 with T was obtained at a recombination fraction (Θ) of 0.00. One mutant T allele was shared with American Bobtails and Pixie-Bobs; both breeds developed more recently in the US. The ability of mutant Brachyury protein to activate transcription of a downstream target was substantially lower than wild-type protein. Collectively, these results suggest that haploinsufficiency of Brachyury is one mechanism underlying variable tail length in domesticated cats.

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Acknowledgments

We are deeply indebted to the community of Manx, American Bobtail, and Japanese Bobtail breeders and the Mann Cat Sanctuary for their assistance and helpful discussion. We also thank Bill Clark and Dawn Reding of Iowa State University for American Bobcat DNA samples. Funding was provided in part by the National Center for Research Resources (R24 RR016094) and research is currently supported by the Office of Research Infrastructure Programs (OD R24OD010928), the Winn Feline Foundation (grant 10-015), and the UC Davis Center for Companion Animal Health George and Phyllis Miller Feline Health Fund (LAL). Research was also supported by grants from the NIAID (AI061061) and the American Cancer Society (RSG-09-045-01-DDC) to ASW.

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Correspondence to Kati J. Buckingham or Michael J. Bamshad.

Electronic supplementary material

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335_2013_9471_MOESM1_ESM.pdf

Supplementary Table 1. Primers used to amplify and sequence coding regions of the feline T gene. Primers with “PCR” in the primer name were used for amplification and primers with “Seq” in the primer name were used for sequencing. Primers marked with $ and # indicate primers with identical sequence that were used in PCR and sequencing, respectively. All primers map to introns and capture the complete coding sequence of the specified exon and the intron–exon boundaries, with the exception of the forward primer for exon 2 (noted with an *), which is exonic and captures only a portion of the coding sequence (PDF 30 kb)

335_2013_9471_MOESM2_ESM.pdf

Supplementary Table 2. Sequences and map positions (Sep 2011 ICGSC Felis_catus 6.2/ felCat5) of the coding exons of the feline T gene. A partial sequence was deposited in the European Nucleotide Archive under accession number HG004538 (http://www.ebi.ac.uk/ena/data/view/HG004538) (PDF 34 kb)

335_2013_9471_MOESM3_ESM.pdf

Supplementary Table 3. Description of tail lengths and mutations in Manx cats with short-tailed cats categorized by specific tail length. (PDF 27 kb)

Supplementary Table 4. Catalog of coding variants in the feline T gene. (PDF 23 kb)

Supplementary Table 5. Summary of tail lengths and mutation counts for all breeds. (PDF 43 kb)

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Supplementary Fig. 1 Delineation of sub-pedigrees used in linkage analysis. To facilitate linkage analysis, the complex pedigrees were divided into six smaller subpedigrees defined by various line colors: Pedigree A1 containing subpedigree 1 (orange), 2 (navy blue), 3 (green), and 4 (pink); Pedigree A1 and A2 containing subpedigree 5 (purple); and Pedigree B containing subpedigree 6 (teal). Individuals connected with black lines were not used in linkage analysis. (EPS 626 kb)

335_2013_9471_MOESM7_ESM.eps

Supplementary Fig. 2. Manx Pedigree B. Extended pedigree illustrating relationships among Manx cats used for linkage and mutation analysis. Squares represent male cats and circles represent female cats. Cats with reduced tail lengths are denoted by varied darkened symbols: rumpy, quarter filled; rumpy-riser, half filled; stumpy, three-quarter filled. Full-tailed cats are indicated with open symbols and patterned symbols denote individuals for whom no phenotypic data were available. Males represented more than once in the pedigree are indicated with a double square labeled with the generation and individual number of the first time they appear in the pedigree. Genotypes for the c.998delT and c.1169delC or c.1199delC T mutants are indicated below each symbol. A “-” indicates the mutation was absent. (EPS 411 kb)

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Buckingham, K.J., McMillin, M.J., Brassil, M.M. et al. Multiple mutant T alleles cause haploinsufficiency of Brachyury and short tails in Manx cats. Mamm Genome 24, 400–408 (2013). https://doi.org/10.1007/s00335-013-9471-1

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Keywords

  • Tail Length
  • Imperforate Anus
  • Short Tail
  • Sacral Spinal Cord
  • Transcriptional Activation Potential