A genome-wide association study of osteochondritis dissecans in the Thoroughbred

Osteochondrosis is a developmental orthopaedic disease that occurs in horses, other livestock species, companion animal species, and humans. The principal aim of this study was to identify quantitative trait loci (QTL) associated with osteochondritis dissecans (OCD) in the Thoroughbred using a genome-wide association study. A secondary objective was to test the effect of previously identified QTL in the current population. Over 300 horses, classified as cases or controls according to clinical findings, were genotyped for the Illumina Equine SNP50 BeadChip. An animal model was first implemented in order to adjust each horse's phenotypic status for average relatedness among horses and other potentially confounding factors which were present in the data. The genome-wide association test was then conducted on the residuals from the animal model. A single SNP on chromosome 3 was found to be associated with OCD at a genome-wide level of significance, as determined by permutation. According to the current sequence annotation, the SNP is located in an intergenic region of the genome. The effects of 24 SNPs, representing QTL previously identified in a sample of Hanoverian Warmblood horses, were tested directly in the animal model. When fitted alongside the significant SNP on ECA3, two of these SNPs were found to be associated with OCD. Confirmation of the putative QTL identified on ECA3 requires validation in an independent sample. The results of this study suggest that a significant challenge faced by equine researchers is the generation of sufficiently large data sets to effectively study complex diseases such as osteochondrosis.     

The identification of SNPs with indeterminate positions using the Equine SNP50 BeadChip

We have used linkage disequilibrium (LD) to identify single nucleotide polymorphisms (SNPs) on the Illumina Equine SNP50 BeadChip, which may be incorrectly positioned on the genome map. A total of 1201 Thoroughbred horses were genotyped using the Illumina Equine SNP50 BeadChip. LD was evaluated in a pairwise fashion between all autosomal SNPs, both within and across chromosomes. Filters were then applied to the data, firstly to identify SNPs that may have been mapped to the wrong chromosome and secondly to identify SNPs that may have been incorrectly positioned within chromosomes. We identified a single SNP on ECA28, which showed low LD with neighbouring SNPs but considerable LD with a group of SNPs on ECA10. Furthermore, a cluster of SNPs on ECA5 showed unusually low LD with surrounding SNPs. A total of 39 SNPs met the criteria for unusual within-chromosome LD. The results of this study indicate that some SNPs may be misplaced. This finding is significant, as misplaced SNPs may lead to difficulties in the application of genomic methods, such as homozygosity mapping, for which SNP order is important.     

Refinement of a quantitative gene locus on equine chromosome 16 responsible for osteochondrosis in Hanoverian warmblood horses

Osteochondrosis (OC) is an inherited developmental disease in young horses most frequently observed in thoroughbreds, trotters, warmblood and coldblood horses. Quantitative trait loci (QTL) for equine OC have been identified in Hanoverian warmblood horses employing a whole genome scan with microsatellites. A QTL on ECA16 reached the genome-wide significance level for hock osteochondrosis dissecans (OCD). The aim of this study was to refine this QTL on ECA16 using an extended marker set of 34 newly developed microsatellites and 15 single nucleotide polymorphisms (SNPs). We used the same 14 paternal half-sib groups as in the above-mentioned whole genome scan. The QTL for OCD in hock joints on ECA16 could be delimited at an interval between 17.60 and 45.18 Mb using multipoint non-parametric linkage analyses. In addition, six microsatellites and one SNP were significantly associated with hock OCD in the QTL region between 24.26 and 42.41 Mb. Furthermore, our analysis revealed a second QTL for fetlock OC between 6.55 and 24.26 Mb on ECA16. This report is a further step towards unravelling the genes underlying QTL for equine OC and towards the development of a marker test for OC in Hanoverian warmblood horses.     

Differences between horse selection based on two forms of osteochondrosis in fetlock

Horses lose potential opportunities because of health problems. Available breeding strategies are not effective enough, probably also because of the different definition used and its genetic usefulness. The aim of the study was to compare the genetic background estimated by the genome-wide association study (GWAS) for osteochondrosis using two different scaling osteochondrosis (OC)/healthy and osteochondrosis dissecans (OCD)/healthy systems for evaluating the disease status of investigated fetlock joints. Two hundred one Warmblood horses trained for performance tests (87 stallions and 114 mares) were phenotyped and genotyped. Four fetlock x-ray images per horse were collected using the RTG Girth HF 80 and Vet Scan ray 3600. The DNA of each horse was genotyped using the BeadChip 70K. To identify SNPs that significantly affect the probability of osteochondrosis, two different methods were applied: the Cochran-Armitage test based on an additive mode of inheritance and logistic regression. The genetic background for osteochondrosis, expressed in the number of SNPs found with significant associations with osteochondrosis, was higher by evaluation in the scale of OCD/healthy horses (16 SNPs on several chromosomes mainly on the ECA1 and ECA10) than OC/healthy (2 SNPs on the ECA15 and one SNP on the ECA10). Detailed definition of osteochondrosis is needed in breeding and in veterinary practice. The genetic background for osteochondrosis and osteochondrosis dissecans seems not the same. Suggestive SNPs could be the candidate markers for osteochondrosis but should be checked on a larger population before usage.     

A genome-wide association study for quantitative trait loci of show-jumping in Hanoverian warmblood horses

Show-jumping is an economically important breeding goal in Hanoverian warmblood horses. The aim of this study was a genome-wide association study (GWAS) for quantitative trait loci (QTL) for show-jumping in Hanoverian warmblood horses, employing the Illumina equine SNP50 Beadchip. For our analyses, we genotyped 115 stallions of the National State stud of Lower Saxony. The show-jumping talent of a horse includes style and ability in free-jumping. To control spurious associations based on population stratification, two different mixed linear animal model (MLM) approaches were employed, besides linear models with fixed effects only and adaptive permutations for correcting multiple testing. Population stratification was explained best in the MLM considering Hanoverian, Thoroughbred, Trakehner and Holsteiner genes and the marker identity-by-state relationship matrix. We identified six QTL for show-jumping on horse chromosomes (ECA) 1, 8, 9 and 26 (-log(10) P-value >5) and further putative QTL with -log(10) P-values of 3-5 on ECA1, 3, 11, 17 and 21. Within six QTL regions, we identified human performance-related genes including PAPSS2 on ECA1, MYL2 on ECA8, TRHR on ECA9 and GABPA on ECA26 and within the putative QTL regions NRAP on ECA1, and TBX4 on ECA11. The results of our GWAS suggest that genes involved in muscle structure, development and metabolism are crucial for elite show-jumping performance. Further studies are required to validate these QTL in larger data sets and further horse populations.     

Associations between candidate gene markers at a quantitative trait locus on equine chromosome 4 responsible for osteochondrosis dissecans in fetlock joints of South German Coldblood horses

A previously accomplished whole-genome scan for osteochondrosis (OC) and OC dissecans (OCD) in South German Coldblood horses using 250 microsatellite markers identified putative quantitative trait loci (QTL). A chromosome-wide significant QTL for fetlock OCD was found on Equus caballus chromosome (ECA) 4q at a relative position of 70.0-73.3 cM. The aim of this study was to analyze associations of single nucleotide polymorphisms (SNPs) in candidate genes for OC in this region. The association analysis included 32 affected and 64 unaffected horses. Three SNPs located in intron 8, intron 9, and 3'-untranslated region (UTR) of the acyloxyacyl hydrolase (AOAH) gene on ECA4q were significantly associated with OCD in fetlock joints. In order to control for systematic environmental and quantitative genetic effects, we employed a linear animal model. The association of the SNP (AJ543065:g.703A>G) in the 3'-UTR of exon 21 was confirmed in the animal model analysis and a significant additive genetic effect for fetlock OCD of 0.42 (P = 0.002) and a dominance effect of -0.32 (P = 0.03) was estimated. This is the first report on a marker in population-wide linkage disequilibrium with equine OCD in fetlock joints.     

A Genome-Wide Association Study Identifies Risk Loci to Equine Recurrent Uveitis in German Warmblood Horses

Equine recurrent uveitis (ERU) is a common eye disease affecting up to 3–15% of the horse population. A genome-wide association study (GWAS) using the Illumina equine SNP50 bead chip was performed to identify loci conferring risk to ERU. The sample included a total of 144 German warmblood horses. A GWAS showed a significant single nucleotide polymorphism (SNP) on horse chromosome (ECA) 20 at 49.3 Mb, with IL-17A and IL-17F being the closest genes. This locus explained a fraction of 23% of the phenotypic variance for ERU. A GWAS taking into account the severity of ERU, revealed a SNP on ECA18 nearby to the crystalline gene cluster CRYGA-CRYGF. For both genomic regions on ECA18 and 20, significantly associated haplotypes containing the genome-wide significant SNPs could be demonstrated. In conclusion, our results are indicative for a genetic component regulating the possible critical role of IL-17A and IL-17F in the pathogenesis of ERU. The associated SNP on ECA18 may be indicative for cataract formation in the course of ERU.     

Genome-wide association study among four horse breeds identifies a common haplotype associated with in vitro CD3+ T cell susceptibility/resistance to equine arteritis virus infection

Previously, we have shown that horses could be divided into susceptible and resistant groups based on an in vitro assay using dual-color flow cytometric analysis of CD3+ T cells infected with equine arteritis virus (EAV). Here, we demonstrate that the differences in in vitro susceptibility of equine CD3+ T lymphocytes to EAV infection have a genetic basis. To investigate the possible hereditary basis for this trait, we conducted a genome-wide association study (GWAS) to compare susceptible and resistant phenotypes. Testing of 267 DNA samples from four horse breeds that had a susceptible or a resistant CD3+ T lymphocyte phenotype using both Illumina Equine SNP50 BeadChip and Sequenom's MassARRAY system identified a common, genetically dominant haplotype associated with the susceptible phenotype in a region of equine chromosome 11 (ECA11), positions 49572804 to 49643932. The presence of a common haplotype indicates that the trait occurred in a common ancestor of all four breeds, suggesting that it may be segregated among other modern horse breeds. Biological pathway analysis revealed several cellular genes within this region of ECA11 encoding proteins associated with virus attachment and entry, cytoskeletal organization, and NF-κB pathways that may be associated with the trait responsible for the in vitro susceptibility/resistance of CD3+ T lymphocytes to EAV infection. The data presented in this study demonstrated a strong association of genetic markers with the trait, representing de facto proof that the trait is under genetic control. To our knowledge, this is the first GWAS of an equine infectious disease and the first GWAS of equine viral arteritis.     

Genetic investigation of equine recurrent uveitis in Appaloosa horses

Equine recurrent uveitis (ERU) is characterized by intraocular inflammation that often leads to blindness in horses. Appaloosas are more likely than any other breed to develop insidious ERU, distinguished by low-grade chronic intraocular inflammation, suggesting a genetic predisposition. Appaloosas are known for their white coat spotting patterns caused by the leopard complex spotting allele (LP) and the modifier PATN1. A marker linked to LP on ECA1 and markers near MHC on ECA20 were previously associated with increased ERU risk. This study aims to further investigate these loci and identify additional genetic risk factors. A GWAS was performed using the Illumina Equine SNP70 BeadChip in 91 horses. Additive mixed model approaches were used to correct for relatedness. Although they do not reach a strict Bonferroni genome-wide significance threshold, two SNPs on ECA1 and one SNP each on ECA12 and ECA29 were among the highest ranking SNPs and thus warranted further analysis (P = 1.20 × 10⁻⁵, P = 5.91 × 10⁻⁶, P = 4.91 × 10⁻⁵, P = 6.46 × 10⁻⁵). In a second cohort (n = 98), only an association with the LP allele on ECA1 was replicated (P = 5.33 × 10⁻⁵). Modeling disease risk with LP, age and additional depigmentation factors (PATN1 genotype and extent of roaning) supports an additive role for LP and suggests an additive role for PATN1. Genotyping for LP and PATN1 may help predict ERU risk (AUC = 0.83). The functional role of LP and PATN1 in ERU development requires further investigation. Testing samples across breeds with leopard complex spotting patterns and a denser set of markers is warranted to further refine the genetic components of ERU.     

Identification of the myostatin locus (MSTN) as having a major effect on optimum racing distance in the Thoroughbred horse in the USA

One hundred and eighty-nine Thoroughbred horses that had won Graded Stakes races in North America were genotyped with the Illumina Equine SNP50 bead chip. Association tests using PLINK to determine whether any SNPs were associated with optimum racing distance (7 furlongs and under compared to 8-10 furlongs) identified a locus on ECA18 that was statistically significant (-log 10 EMP2=1.63) at the genome-wide level following permutation analysis (10,000 permutations). Bioinformatic analysis revealed that the two ECA18 SNPs with the highest statistical significance spanned the MSTN (myostatin) locus. Mutations in myostatin in several mammalian species have been associated with increased muscling, with a preferential increase in fast glycolytic type IIB fibres, which would increase power potential. Thoroughbred horses that race over sprint distances, which are 5-7 furlongs, are often characterized by impressive hind quarter musculature, strongly suggesting that the association observed between the ECA18 SNPs and optimum race distance is mediated through MSTN.     

Genome-wide linkage and association analysis identifies major gene Loci for guttural pouch tympany in arabian and german warmblood horses

Equine guttural pouch tympany (GPT) is a hereditary condition affecting foals in their first months of life. Complex segregation analyses in Arabian and German warmblood horses showed the involvement of a major gene as very likely. Genome-wide linkage and association analyses including a high density marker set of single nucleotide polymorphisms (SNPs) were performed to map the genomic region harbouring the potential major gene for GPT. A total of 85 Arabian and 373 German warmblood horses were genotyped on the Illumina equine SNP50 beadchip. Non-parametric multipoint linkage analyses showed genome-wide significance on horse chromosomes (ECA) 3 for German warmblood at 16-26 Mb and 34-55 Mb and for Arabian on ECA15 at 64-65 Mb. Genome-wide association analyses confirmed the linked regions for both breeds. In Arabian, genome-wide association was detected at 64 Mb within the region with the highest linkage peak on ECA15. For German warmblood, signals for genome-wide association were close to the peak region of linkage at 52 Mb on ECA3. The odds ratio for the SNP with the highest genome-wide association was 0.12 for the Arabian. In conclusion, the refinement of the regions with the Illumina equine SNP50 beadchip is an important step to unravel the responsible mutations for GPT.     

Equine developmental orthopaedic diseases – a genome‐wide association study of first phalanx plantar osteochondral fragments in Standardbred trotters

Palmar/plantar osteochondral fragments (POF) in fetlock joints commonly affect and influence the athletic performance of horses. In this study, we used the Equine SNP50 BeadChip^® to perform a genome‐wide association study of metatarsophalangeal POF in 176 Norwegian Standardbred trotter yearlings. Putative quantitative trait loci (QTL) for medial and/or lateral POF, and medial POF only were identified on ECA1, 2, 7, 9 and 31, whereas for lateral POF, only on ECA7, 11, 27 and X. The moderate number of QTL evidences a complex inheritance and suggests various genes controlling POF development in medial and lateral locations.     

A Non-Synonymous HMGA2 Variant Decreases Height in Shetland Ponies and Other Small Horses

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.     

Mapping quantitative trait loci for osteochondrosis in fetlock and hock joints and palmar/plantar osseus fragments in fetlock joints of South German Coldblood horses

The aim of this study was to identify quantitative trait loci (QTL) for osteochondrosis (OC) and palmar/plantar osseous fragments (POF) in fetlock joints in a whole-genome scan of 219 South German Coldblood horses. Symptoms of OC and POF were checked by radiography in 117 South German Coldblood horses at a mean age of 17 months. The radiographic examination comprised the fetlock and hock joints of all limbs. The genome scan included 157 polymorphic microsatellite markers. All microsatellite markers were equally spaced over the 31 autosomes and the X chromosome, with an average distance of 17.7 cM and a mean polymorphism information content (PIC) of 63%. Sixteen chromosomes harbouring putative QTL regions were further investigated by genotyping the animals with 93 additional markers. QTL that had chromosome-wide significance by non-parametric Z-means and LOD scores were found on 10 chromosomes. This included seven QTL for fetlock OC and one QTL on ECA18 associated with hock OC and fetlock OC. Significant QTL for POF in fetlock joints were located on equine chromosomes 1, 4, 8, 12 and 18. This genome scan is an important step towards the identification of genes responsible for OC in horses.     

Whole-genome scan for guttural pouch tympany in Arabian and German warmblood horses

Equine guttural pouch tympany (GPT) is a hereditary disease in foals of several breeds, including thoroughbreds, Arabian, Quarter and warmblood horses. We performed a whole-genome scan for GPT in 143 horses from five Arabian and five German warmblood families and genotyped 257 microsatellites. Chromosome-wide significant linkage was detected on ECA2 and ECA15 using multipoint non-parametric linkage analyses. Analyses stratified by sex revealed chromosome-wide significant linkage on ECA2 for fillies and chromosome-wide significant linkage on ECA15 for colts. For Arabian colts, the quantitative trait locus (QTL) on ECA15 was genome-wide significant. Haplotypes including two to four microsatellites within the QTL on ECA2 and 15 in fillies and colts, respectively, were significantly associated with GPT for both breeds. Thus, our analysis indicated sex-specific QTL, a fact which is in agreement with a two- to fourfold higher incidence of GPT in females. This is the first report of QTL for equine GPT and a first step towards identifying genes responsible for GPT.     

A genome-wide association study of production traits in a commercial population of Large White pigs: evidence of haplotypes affecting meat quality

Background

Numerous quantitative trait loci (QTL) have been detected in pigs over the past 20 years using microsatellite markers. However, due to the low density of these markers, the accuracy of QTL location has generally been poor. Since 2009, the dense genome coverage provided by the Illumina PorcineSNP60 BeadChip has made it possible to more accurately map QTL using genome-wide association studies (GWAS). Our objective was to perform high-density GWAS in order to identify genomic regions and corresponding haplotypes associated with production traits in a French Large White population of pigs.

Methods

Animals (385 Large White pigs from 106 sires) were genotyped using the PorcineSNP60 BeadChip and evaluated for 19 traits related to feed intake, growth, carcass composition and meat quality. Of the 64 432 SNPs on the chip, 44 412 were used for GWAS with an animal mixed model that included a regression coefficient for the tested SNPs and a genomic kinship matrix. SNP haplotype effects in QTL regions were then tested for association with phenotypes following phase reconstruction based on the Sscrofa10.2 pig genome assembly.

Results

Twenty-three QTL regions were identified on autosomes and their effects ranged from 0.25 to 0.75 phenotypic standard deviation units for feed intake and feed efficiency (four QTL), carcass (12 QTL) and meat quality traits (seven QTL). The 10 most significant QTL regions had effects on carcass (chromosomes 7, 10, 16, 17 and 18) and meat quality traits (two regions on chromosome 1 and one region on chromosomes 8, 9 and 13). Thirteen of the 23 QTL regions had not been previously described. A haplotype block of 183 kb on chromosome 1 (six SNPs) was identified and displayed three distinct haplotypes with significant (0.0001 < P < 0.03) associations with all evaluated meat quality traits.

Conclusions

GWAS analyses with the PorcineSNP60 BeadChip enabled the detection of 23 QTL regions that affect feed consumption, carcass and meat quality traits in a LW population, of which 13 were novel QTL. The proportionally larger number of QTL found for meat quality traits suggests a specific opportunity for improving these traits in the pig by genomic selection.     

Fine mapping of a quantitative trait locus for osteochondrosis on horse chromosome 2

In this study, we refine a quantitative trait locus for equine osteochondrosis (OC) on horse chromosome (ECA) 2 to a genome-wide significant interval at 20.08-30.94 Mb. The marker set contained 27 newly developed microsatellites equidistantly distributed over ECA2 and 44 nucleotide polymorphisms, located in 16 positional candidate genes for OC. Genotyping was performed in 211 Hanoverian horses from 14 paternal half-sib groups. A NCDN-associated SNP and haplotype were significantly associated with OC in fetlock and/or hock joints. This study is a further step towards the identification of genes responsible for OC in horses.     

Refinement of quantitative trait loci on equine chromosome 10 for radiological signs of navicular disease in Hanoverian warmblood horses

Navicular disease is characterized by a progressive degenerative alteration of the equine podotrochlea. In this study, we refined a previously identified quantitative trait locus (QTL) on horse chromosome 10 for the abnormal development of canales sesamoidales (DCS) of the navicular bone in Hanoverian warmblood horses. Genotyping was done in 192 Hanoverian warmblood horses from 17 paternal half-sib groups. The whole marker set comprised 45 markers including seven newly developed microsatellites and 13 single nucleotide polymorphisms (SNPs) within positional candidate genes. Chromosome-wide significant QTL were confirmed and refined for DCS on horse chromosome (ECA) 10 at 0.16-2.70 Mb and at 14.45-36.37 Mb. Nine microsatellites and three SNP markers reached the highest multipoint Zmeans and LOD scores at 19.34-20.38 Mb and at 23.17-30.73 Mb with genome-wide error probabilities of P<0.05. In addition, a significant association of a SNP within VSTM1 and a significant haplotype-trait association within IRF3 could be shown. These results support a possible role of the candidate genes VSTM1 and IRF3 within the QTL on ECA10 for DCS. This study is a further step towards the identification of the genes responsible for navicular disease in Hanoverian warmblood horses.     

Refinement of a quantitative trait locus on equine chromosome 5 responsible for fetlock osteochondrosis in Hanoverian warmblood horses

In this report, we provide 29 new informative microsatellites distributed over a region of 21 Mb on horse chromosome (ECA) 5 and refine a quantitative trait locus (QTL) for fetlock osteochondrosis dissecans (OCD) to a genome-wide significant interval between 78.03 and 90.23 Mb on ECA5. Genotyping was performed in 211 Hanoverian warmblood horses from 14 paternal half-sib groups. Within this OCD-QTL, collagen type XXIV alpha 1 was identified as a potential functional candidate gene for equine osteochondrosis. This report is a further step towards unravelling the genes that cause equine osteochondrosis.     

Mapping of equine potassium chloride co-transporter (SLC12A4) and amino acid transporter (SLC7A10) and preliminary studies on associations between SNPs from SLC12A4, SLC7A10 and SLC7A9 and osmotic fragility of erythrocytes

Consensus DNA sequences from human, mouse and/or rat were used to design oligonucleotide primers for equine homologues of exons 16, 17 and 20-23 of potassium chloride co-transporter (SLC12A4) and exons 10, 11 and 3, 4, respectively, for two amino acid transporters (SLC7A10 and SLC7A9). DNA sequences of the PCR products showed high sequence identity to these regions. Equine BAC clones were obtained for SLC12A4 and SLC7A10 and mapped to equine chromosomes ECA3p13 and ECA10p15, respectively, by fluorescence in situ hybridization (FISH). Several single nucleotide polymorphisms (SNP) were found. Substitutions of A/G were found within exon 17 of SLC12A4, within intron 11 of SLC7A10 and within intron 3 of SLC7A9. The SNP associated with SLC7A10 and SLC7A9 were sufficiently polymorphic to investigate associations with erythrocyte fragility among a group of 20 thoroughbred horses. A non-parametric rank-sum test showed a weak association between erythrocyte fragility and the SNP associated with SLC7A10 (P < 0.05).