Skip to main content

Whole-genome sequencing reveals a potential causal mutation for dwarfism in the Miniature Shetland pony

Abstract

The Miniature Shetland pony represents a horse breed with an extremely small body size. Clinical examination of a dwarf Miniature Shetland pony revealed a lowered size at the withers, malformed skull and brachygnathia superior. Computed tomography (CT) showed a shortened maxilla and a cleft of the hard and soft palate which protruded into the nasal passage leading to breathing difficulties. Pathological examination confirmed these findings but did not reveal histopathological signs of premature ossification in limbs or cranial sutures. Whole-genome sequencing of this dwarf Miniature Shetland pony and comparative sequence analysis using 26 reference equids from NCBI Sequence Read Archive revealed three probably damaging missense variants which could be exclusively found in the affected foal. Validation of these three missense mutations in 159 control horses from different horse breeds and five donkeys revealed only the aggrecan (ACAN)-associated g.94370258G>C variant as homozygous wild-type in all control samples. The dwarf Miniature Shetland pony had the homozygous mutant genotype C/C of the ACAN:g.94370258G>C variant and the normal parents were heterozygous G/C. An unaffected full sib and 3/5 unaffected half-sibs were heterozygous G/C for the ACAN:g.94370258G>C variant. In summary, we could demonstrate a dwarf phenotype in a miniature pony breed perfectly associated with a missense mutation within the ACAN gene.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

References

  • Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P, Kondrashov AS, Sunyaev SR (2010) A method and server for predicting damaging missense mutations. Nat Methods 7:248–249

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Andrews S (2010) FastQC: A quality control tool for high throughput sequence data. In Reference Source http://www.bioinformatics.babraham.ac.uk/projects/fastqc/

  • Beever JE, Smit MA, Meyers SN, Hadfield TS, Bottema C, Albretsen J, Cockett NE (2006) A single-base change in the tyrosine kinase II domain of ovine FGFR3 causes hereditary chondrodysplasia in sheep. Anim Genet 37:66–71

    CAS  Article  PubMed  Google Scholar 

  • Bingel SA, Sande RD (1982) Chondrodysplasia in the Norwegian Elkhound. Am J Pathol 107:219–229

    CAS  PubMed  PubMed Central  Google Scholar 

  • Bundschu K, Knobeloch K-P, Ullrich M, Schinke T, Amling M, Engelhardt CM, Renné T, Walter U, Schuh K (2005) Gene disruption of Spred-2 causes dwarfism. J Biol Chem 280:28572–28580

    CAS  Article  PubMed  Google Scholar 

  • Cavanagh JA, Tammen I, Windsor PA, Bateman JF, Savarirayan R, Nicholas FW, Raadsma HW (2007) Bulldog dwarfism in Dexter cattle is caused by mutations in ACAN. Mamm Genome 18:808–814

    CAS  Article  PubMed  Google Scholar 

  • Cingolani P, Platts A, Wang le L, Coon M, Nguyen T, Wang L, Land SJ, Lu X, Ruden DM (2012) A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly (Austin) 6:80–92

    CAS  Article  Google Scholar 

  • Drickamer K, Taylor ME (1993) Biology of animal lectins. Annu Rev Cell Biol 9:237–264

    CAS  Article  PubMed  Google Scholar 

  • Eberth JE (2013) Chondrodysplasia-Like dwarfism in the Miniature Horse. Kentucky. University of Kentucky, Lexington

    Google Scholar 

  • Eberth J, Swerczak T, Bailey E (2009) Investigation of dwarfism Among Miniature Horses using the Illumina Horse SNP50 Bead Chip. J Equine Veterinary Sci 29:315

    Article  Google Scholar 

  • Eigenmann J (1981) Diagnosis and treatment of dwarfism in a German Shepherd dog. J Am Animal Hosp Assoc (USA) 17:798–804

    Google Scholar 

  • Heinegård D, Oldberg A (1989) Structure and biology of cartilage and bone matrix noncollagenous macromolecules. FASEB J 3:2042–2051

    PubMed  Google Scholar 

  • Iwata T, Chen L, Li C, Ovchinnikov DA, Behringer RR, Francomano CA, Deng CX (2000) A neonatal lethal mutation in FGFR3 uncouples proliferation and differentiation of growth plate chondrocytes in embryos. Hum Mol Genet 9:1603–1613

    CAS  Article  PubMed  Google Scholar 

  • Koparir A, Karatas OF, Yuceturk B, Yuksel B, Bayrak AO, Gerdan OF, Sagiroglu MS, Gezdirici A, Kirimtay K, Selcuk E (2015) Novel POC1A mutation in primordial dwarfism reveals new insights for centriole biogenesis. Hum Mol Genet 24:5378–5387

    CAS  Article  PubMed  Google Scholar 

  • Kumar P, Henikoff S, Ng PC (2009) Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nat Protoc 4:1073–1081

    CAS  Article  PubMed  Google Scholar 

  • Legeai-Mallet L, Benoist-Lasselin C, Munnich A, Bonaventure J (2004) Overexpression of FGFR3, Stat1, Stat5 and p21Cip1 correlates with phenotypic severity and defective chondrocyte differentiation in FGFR3-related chondrodysplasias. Bone 34:26–36

    CAS  Article  PubMed  Google Scholar 

  • Li H, Durbin R (2010) Fast and accurate long-read alignment with Burrows–Wheeler transform. Bioinformatics 26:589–595

    Article  PubMed  PubMed Central  Google Scholar 

  • Li H, Schwartz NB, Vertel BM (1993) cDNA cloning of chick cartilage chondroitin sulfate (aggrecan) core protein and identification of a stop codon in the aggrecan gene associated with the chondrodystrophy, nanomelia. J Biol Chem 268:23504–23511

    CAS  PubMed  Google Scholar 

  • Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R, Genome Project Data Processing S (2009) The sequence Alignment/Map format and SAM tools. Bioinformatics 25:2078–2079

    Article  PubMed  PubMed Central  Google Scholar 

  • McLaren W, Pritchard B, Rios D, Chen Y, Flicek P, Cunningham F (2010) Deriving the consequences of genomic variants with the Ensembl API and SNP effect predictor. Bioinformatics 26:2069–2070

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Metzger J, Schrimpf R, Philipp U, Distl O (2013) Expression levels of LCORL are associated with body size in horses. PLoS One 8:1–9

    Google Scholar 

  • Metzger J, Karwath M, Tonda R, Beltran S, Agueda L, Gut M, Gut IG, Distl O (2015) Runs of homozygosity reveal signatures of positive selection for reproduction traits in breed and non-breed horses. BMC Genomics 16:764

    Article  PubMed  PubMed Central  Google Scholar 

  • Meyers V, Aguirre G, Patterson D (1983) Short-limbed dwarfism and ocular defects in the Samoyed dog. J Am Vet Med Assoc 183:975–979

    CAS  PubMed  Google Scholar 

  • Orr N, Back W, Gu J, Leegwater P, Govindarajan P, Conroy J, Ducro B, Van Arendonk JA, MacHugh DE, Ennis S, Hill EW, Brama PA (2010) Genome-wide SNP association-based localization of a dwarfism gene in Friesian dwarf horses. Anim Genet 41(Suppl 2):2–7

    CAS  Article  PubMed  Google Scholar 

  • Quinlan AR, Hall IM (2010) BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26:841–842

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Salvatori R, Hayashida CY, Aguiar-Oliveira MH, Phillips JA III, Souza AH, Gondo RrG, Toledo SP, Conceicão MM, Prince M, Maheshwari HG (1999) Familial dwarfism due to a novel mutation of the growth hormone-releasing hormone receptor gene 1. J Clin Endocrinol Metab 84:917–923

    CAS  PubMed  Google Scholar 

  • Schurink A, Hellinga I, Leegwater P, Back W, Bastiaansen J, Ducro B (2014) Genome-Wide Association Study Leads to a DNA Assay for dwarfism in the Friesian Horse Population. In 10th World Congress on Genetics Applied to Livestock Production: Asas

  • Schwartz NB, Domowicz M (2002) Chondrodysplasias due to proteoglycan defects. Glycobiology 12:57R–68R

    CAS  Article  PubMed  Google Scholar 

  • Smith DC (2016) Book of Miniature Horses: A Guide to Selecting, Caring. Rowman & Littlefield, Lanham

    Google Scholar 

  • Stattin EL, Wiklund F, Lindblom K, Onnerfjord P, Jonsson BA, Tegner Y, Sasaki T, Struglics A, Lohmander S, Dahl N, Heinegard D, Aspberg A (2010) A missense mutation in the aggrecan C-type lectin domain disrupts extracellular matrix interactions and causes dominant familial osteochondritis dissecans. Am J Hum Genet 86:126–137

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Tompson SW, Merriman B, Funari VA, Fresquet M, Lachman RS, Rimoin DL, Nelson SF, Briggs MD, Cohn DH, Krakow D (2009) A recessive skeletal dysplasia, SEMD aggrecan type, results from a missense mutation affecting the C-type lectin domain of aggrecan. Am J Hum Genet 84:72–79

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Walker S (2007) Monitoring zum Wachstum und zu Gliedmaßenveränderungen von Junghengsten in Schleswig–Holstein. Christian-Albrechts-Universität zu Kiel

    Google Scholar 

  • Watanabe H, Kimata K, Line S, Strong D, Gao LY, Kozak CA, Yamada Y (1994) Mouse cartilage matrix deficiency (cmd) caused by a 7 bp deletion in the aggrecan gene. Nat Genet 7:154–157

    CAS  Article  PubMed  Google Scholar 

  • Watanabe H, Nakata K, Kimata K, Nakanishi I, Yamada Y (1997) Dwarfism and age-associated spinal degeneration of heterozygote cmd mice defective in aggrecan. Proc Natl Acad Sci USA 94:6943–6947

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Yang BB, Zhang Y, Cao L, Yang BL (1998) Aggrecan and link protein affect cell adhesion to culture plates and to type II collagen. Matrix Biol 16:541–561

    CAS  Article  PubMed  Google Scholar 

Download references

Acknowledgements

The authors are grateful to all horse breeders donating samples and providing data for this research project. The authors thank Dr. R. Geffers and S. Buhju at the Helmholtz Centre for Infection Research in Braunschweig for their support in library preparation. We also thank J. Wrede for his help in data analysis and M. Drabert for his support in DNA isolation.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Ottmar Distl.

Electronic supplementary material

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Metzger, J., Gast, A.C., Schrimpf, R. et al. Whole-genome sequencing reveals a potential causal mutation for dwarfism in the Miniature Shetland pony. Mamm Genome 28, 143–151 (2017). https://doi.org/10.1007/s00335-016-9673-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00335-016-9673-4

Keywords

  • Osteochondritis Dissecans
  • Epiphyseal Plate
  • Dwarf Phenotype
  • ACAN Gene
  • NCBI Sequence Read Archive