A Non-Synonymous HMGA2 Variant Decreases Height in Shetland Ponies and Other Small Horses.
ABSTRACT: The identification of quantitative trait loci (QTL) such as height and their underlying causative variants is still challenging and often requires large sample sizes. In humans hundreds of loci with small effects control the heritable portion of height variability. In domestic animals, typically only a few loci with comparatively large effects explain a major fraction of the heritability. We investigated height at withers in Shetland ponies and mapped a QTL to ECA 6 by genome-wide association (GWAS) using a small cohort of only 48 animals and the Illumina equine SNP70 BeadChip. Fine-mapping revealed a shared haplotype block of 793 kb in small Shetland ponies. The HMGA2 gene, known to be associated with height in horses and many other species, was located in the associated haplotype. After closing a gap in the equine reference genome we identified a non-synonymous variant in the first exon of HMGA2 in small Shetland ponies. The variant was predicted to affect the functionally important first AT-hook DNA binding domain of the HMGA2 protein (c.83G>A; p.G28E). We assessed the functional impact and found impaired DNA binding of a peptide with the mutant sequence in an electrophoretic mobility shift assay. This suggests that the HMGA2 variant also affects DNA binding in vivo and thus leads to reduced growth and a smaller stature in Shetland ponies. The identified HMGA2 variant also segregates in several other pony breeds but was not found in regular-sized horse breeds. We therefore conclude that we identified a quantitative trait nucleotide for height in horses.
Project description:BACKGROUND:Miniature size in horses represents an extreme reduction of withers height that originated after domestication. In some breeds, it is a highly desired trait representing a breed- or subtype-specific feature. The genomic changes that emerged due to strong-targeted selection towards this distinct type remain unclear. RESULTS:Comparisons of whole-genome sequencing data from two Miniature Shetland ponies and one standard-sized Shetland pony, performed to elucidate genetic determinants for miniature size, revealed four synergistic variants, limiting withers height to 34.25 in. (87 cm). Runs of homozygosity regions were detected spanning these four variants in both the Miniature Shetland ponies and the standard-sized Shetland pony. They were shown to be characteristic of the Shetland pony breed, resulting in a miniature type under specific genotypic combinations. These four genetic variants explained 72% of the size variation among Shetland ponies and related breeds. The length of the homozygous regions indicate that they arose over 1000 years ago. In addition, a copy number variant was identified in DIAPH3 harboring a loss exclusively in ponies and donkeys and thus representing a potential height-associated variant. CONCLUSION:This study reveals main drivers for miniature size in horses identified in whole genome data and thus provides relevant candidate genes for extremely short stature in mammals.
Project description:BACKGROUND:Ponies are highly susceptible to metabolic derangements including hyperinsulinemia, insulin resistance, and adiposity. HYPOTHESIS/OBJECTIVES:Genetic loci affecting height in ponies have pleiotropic effects on metabolic pathways and increase the susceptibility to equine metabolic syndrome (EMS). ANIMALS:Two hundred ninety-four Welsh ponies and 529 horses. METHODS:Retrospective study of horses phenotyped for metabolic traits. Correlations between height and metabolic traits were assessed by Pearson's correlation coefficients. Complementary genome-wide analysis methods were used to identify a region of interest (ROI) for height and metabolic traits, determine the fraction of heritability contributed by the ROI, and identify candidate genes. RESULTS:There was an inverse relationship between height and baseline insulin (-0.26) in ponies. Genomic signature of selection and association analyses for both height and insulin identified the same ~1.3 megabase region on chromosome 6 that contained a shared ancestral haplotype between these traits. The ROI contributed ~40% of the heritability for height and ~20% of the heritability for insulin. High-mobility group AT-hook 2 was identified as a candidate gene, and Sanger sequencing detected a c.83G>A (p.G28E) variant associated with height in Shetland ponies. In our cohort of ponies, the A allele had a frequency of 0.76, was strongly correlated with height (-0.75), and was low to moderately correlated with metabolic traits including: insulin (0.32), insulin after an oral sugar test (0.25), non-esterified fatty acids (0.19), and triglyceride (0.22) concentrations. CONCLUSIONS AND CLINICAL IMPORTANCE:These data have important implications for identifying individuals at risk for EMS.
Project description:<h4>Background</h4>Much of the shape variation found in animals is based on allometry and heterochrony. Horses represent an excellent model to investigate patterns of size-shape variation among breeds that were intentionally bred for extreme small and large sizes.<h4>Methods</h4>We tested whether ponies (wither height < 148 cm) have a diverging size-shape relationship in skull shape as compared to regular-sized horse breeds (wither height > 148 cm, here-after called horses) during ontogenetic growth. We used a dataset of 194 specimens from 25 horse and 13 pony breeds, two of which are miniature breeds (wither height < 96.5 cm)-Falabella, Shetland. We applied three-dimensional geometric morphometrics, linear measurements, and multivariate analyses (Procrustes ANOVAs) to quantitatively examine and compare the ontogenetic trajectories between pony and horse breeds with an emphasis on the miniature breeds as an extreme case of artificial selection on size. Additionally, we tested for juvenile characteristics in adult horse and miniature breeds that could resemble "paedomorphosis"-retention of juvenile characteristics in adult stage; e.g. large eyes, large braincase-to-face-relationship, and large head-to-body relationship.<h4>Results</h4>Allometric regression of size on shape revealed that 42% of shape variation could be explained by variation in size in all breeds. The ontogenetic trajectories of ponies and horses vary in slope and therefore in rate of change per unit size, and length. The differences in trajectory lengths and slopes result in ponies having a similar skull shape in an older age stage than horses of the same size in a younger age stage. This pattern could cause the generally perceived "paedomorphic" appearance of ponies. Miniature breeds have larger heads in relation to wither height compared to horses, a non-paedomorphic feature in horses specifically. Also, rostra (faces) are longer in adult individuals than in juveniles across all kinds of breeds. This pattern can be explained by the long-face hypothesis for grazing ungulates and could possibly be caused by the mismatch of selection by humans for shorter rostra and the dentition of ruminants.<h4>Conclusions</h4>Miniature breed specimens do not exhibit any of the classical mammalian "paedomorphic" features (large orbits, large heads), except for the adult Falabella that has enlarged orbits, possibly because they are herbivorous ungulates that are affected by functional and metabolic constraints related to low nutrient-food consumption. Instead ponies, including miniature breeds, have faster and shorter ontogenetic growth compared to horses, resulting in adult pony skulls looking in part like juvenile horse skulls.
Project description:Mushroom is a unique coat color phenotype in Shetland Ponies characterized by the dilution of the chestnut coat color to a sepia tone and is hypothesized to be a recessive trait. A genome wide association study (GWAS), utilizing the Affymetrix 670K array (MNEc670k) and a single locus mixed linear model analysis (EMMAX), identified a locus on ECA7 for further investigation (Pcorrected = 2.08 × 10-10). This locus contained a 3 Mb run of homozygosity in the 12 mushroom ponies tested. Analysis of high throughput Illumina sequencing data from one mushroom Shetland pony compared to 87 genomes from horses of various breeds, uncovered a frameshift variant, p.Asp201fs, in the MFSD12 gene encoding the major facilitator superfamily domain containing 12 protein. This variant was perfectly concordant with phenotype in 96 Shetland Ponies (P = 1.15 × 10-22), was identified in the closely related Miniature Horse for which the mushroom phenotype is suspected to occur (fmu = 0.02), and was absent in 252 individuals from seven additional breeds not reported to have the mushroom phenotype. MFSD12 is highly expressed in melanocytes and variants in this gene in humans, mice, and dogs impact pigmentation. Given the role of MFSD12 in melanogenesis, we propose that p.Asp201fs is causal for the dilution observed in mushroom ponies.
Project description:Fatty acids, as key components of cellular membranes and complex lipids, may play a central role in endocrine signalling and the function of adipose tissue and liver. Thus, the lipid fatty acid composition may play a role in health status in the equine. This study aimed to investigate the fatty acid composition of different tissues and liver lipid classes by comparing Warmblood horses and Shetland ponies under defined conditions. We hypothesized that ponies show different lipid patterns than horses in adipose tissue, liver and plasma. Six Warmblood horses and six Shetland ponies were housed and fed under identical conditions. Tissue and blood sampling were performed following a standardized protocol. A one-step lipid extraction, methylation and trans-esterification method with subsequent gas chromatography was used to analyse the total lipid content and fatty acid profile of retroperitoneal, mesocolon and subcutaneous adipose tissue, liver and plasma. Fatty acids were grouped according to their degree of saturation and their conjugated double bond into the respective lipid classes. In the adipose tissues, saturated fatty acids (SFAs) and n-9 monounsaturated fatty acids (n-9 MUFAs) were most present in ponies and horses. N-6 polyunsaturated fatty acids (n-6 PUFAs), followed by SFAs, were most frequently found in liver tissue and plasma in all animals. Horses, in comparison to ponies, had significantly higher n-6 PUFA levels in all tissues and plasma. In liver tissue, horses had significantly lower hepatic iso-branched-chain fatty acids (iso-BCFAs) than ponies. The hepatic fatty acid composition of selected lipid classes was different between horses and ponies. In the polar PL fraction, horses had low n-9 MUFA and n-3 PUFA contents but higher n-6 PUFA contents than ponies. Furthermore, iso-BCFAs are absent in several hepatic lipid fractions of horses but not ponies. The differences in fatty acid lipid classes between horses and ponies provide key information on the species- and location-specific regulation of FA metabolism, thus affecting health status such as inflammatory responses.
Project description:Background:Non-alcoholic fatty liver disease is known as determining part of human obesity. The impact of body weight (BW) gain on liver metabolism has not been extensively investigated yet. Objectives:To investigate hepatic alterations caused by increasing BW in ponies and horses. Animals:A total of 19 non-obese equines (10 Shetland ponies, geldings; nine Warmblood horses, geldings). Methods:Animals received 200% of their metabolizable maintenance energy requirements for 2 years. Serum alkaline phosphatase, glutamate dehydrogenase (GLDH), aspartate aminotransferase (AST), and gamma-glutamyl transferase activities and bile acids were analyzed several times during 2 years of hypercaloric diet. Hepatic lipid content and hepatic levels of the interleukin (IL)-6, tumor necrosis factor ? (TNF?), cluster of differentiation (CD) 68, IL-1?, lipoprotein lipase (LPL), fatty acid-binding protein 1, chemerin and nuclear factor-?B mRNAs were assessed at the start of the study and after 1 and 2 years of excess energy intake. Results:The mean (±SD) BW gain recorded during 2 years of excess energy intake was 29.9 ± 19.4% for ponies and 17 ± 6.74% for horses. The hepatic lipid content was not profoundly affected by increasing BW. Levels of the IL-6, TNF?, CD68 and IL-1? mRNAs did not change during BW gain. Levels of the chemerin mRNA increased significantly in both breeds (ponies: P = 0.02; horses: P = 0.02) in response to BW gain. Significant differences in serum GLDH and AST activities, serum bile acid concentrations and hepatic levels of the LPL mRNA were observed between ponies and horses at the end of the study. Conclusions:Chemerin might represent an interesting marker for future equine obesity research. Interestingly, steatosis caused by increasing BW may occur later in the development of obesity in equines than in humans. Additionally, the hepatic metabolism exhibits differences between ponies and horses, which may explain in part the greater susceptibility of ponies to obesity-associated metabolic dysregulations.
Project description:Equine metabolic syndrome (EMS) is a complex trait for which few genetic studies have been published. Our study objectives were to perform within breed genome-wide association analyses (GWA) to identify associated loci in two high-risk breeds, coupled with meta-analysis to identify shared and unique loci between breeds. GWA for 12 EMS traits identified 303 and 142 associated genomic regions in 264 Welsh ponies and 286 Morgan horses, respectively. Meta-analysis demonstrated that 65 GWA regions were shared across breeds. Region boundaries were defined based on a fixed-size or the breakdown of linkage disequilibrium, and prioritized if they were: shared between breeds or across traits (high priority), identified in a single GWA cohort (medium priority), or shared across traits with no SNPs reaching genome-wide significance (low priority), resulting in 56 high, 26 medium, and seven low priority regions including 1853 candidate genes in the Welsh ponies; and 39 high, eight medium, and nine low priority regions including 1167 candidate genes in the Morgans. The prioritized regions contained protein-coding genes which were functionally enriched for pathways associated with inflammation, glucose metabolism, or lipid metabolism. These data demonstrate that EMS is a polygenic trait with breed-specific risk alleles as well as those shared across breeds.
Project description:Obesity is a major health concern in many domesticated equids animals since it is related to metabolic abnormalities such as insulin dysregulation, hyperlipidaemia or laminitis. Ponies especially are known as "easy keepers" and are often affected by obesity and its related metabolic disorders. Research in the last decade indicated that the intestinal microbiota may play an important role in the development of obesity, at least in humans. Therefore, the objective of our study was to characterize changes in the faecal microbiota during a two-year weight gain programme which compared ponies and warmblood horses. For this purpose, 10 Shetland ponies and ten warmblood horses were fed a ration which provided 200% of their maintenance energy requirement over two years. Feed intake, body weight, body condition and cresty neck score were recorded weekly. At three standardized time points faecal samples were collected to characterize the faecal microbiota and its fermentation products such as short chain fatty acids and lactate. Next generation sequencing was used for the analysis of the faecal microbiota. During body weight gain the richness of the faecal microbiota decreased in ponies. Besides changes in the phylum Firmicutes in ponies that were already described in human studies, we found a decrease of the phylum Fibrobacteres in horses and an increase of the phylum Actinobacteria. We were also able to show that the phylum Fibrobacteres is more common in the microbiota of horses than in the microbiota of ponies. Therefore, the fibrolytic phylum Fibrobacteres seems to be an interesting phylum in the equine microbiota that should receive more attention in future studies.