Journal of Heredity Advance Access originally published online on January 27, 2008
Journal of Heredity 2008 99(2):125-129; doi:10.1093/jhered/esm106
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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
From the Institute for Animal Breeding and Genetics, University of Veterinary Medicine Hannover, Foundation, Bünteweg 17p, 30559 Hannover, Germany (Wittwer, Dierks, Hamann, and Distl)
Address correspondence to O. Distl at the address above, or e-mail: ottmar.distl{at}tiho-hannover.de.
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.
Osteochondrosis (OC) belongs to those diseases of the locomotory system frequently detected radiographically in young horses (Jeffcott 1991; Stock et al. 2005; Wittwer et al. 2006, Wittwer, Hamann, et al. 2007). Disturbed differentiation and maturation of growing cartilage leads to alterations of the joints, where cartilage flaps, osseous fragments, and synovial effusions are the most common signs of OC (Trotter and McIlwraith 1981; Jeffcott and Henson 1998). Osteochondral fragments (joint mice, chips, and corpora libera) are specific signs of OC, and this condition characterizes the underlying osteochondrotic disease as OC dissecans (OCD). In addition to OC and OCD that manifests at specific predilection sites of the fetlock, hock, and stifle joints, plantar/palmar osteochondral fragments (POFs) are frequently observed in fetlock joints of trotters and coldblood horses (Philipsson et al. 1993, Wittwer et al. 2006). In South German Coldblood (SGC) horses POFs were phenotypically and genetically associated with OC and maybe thus part of the OC complex (Wittwer, Hamann, et al. 2007).
A whole-genome scan for OC, OCD, and POFs in 216 SGC horses using 250 polymorphic microsatellite markers was performed by Wittwer, Löhring, et al. (2007). In total, 17 putative QTL located on 17 equine chromosomes were found for OC or OCD in fetlock and/or hock joints and POFs in fetlock joints. Significant effects for OC in fetlock and/or hock joints were shown for markers on 7 chromosomes. There were 9 QTL significant for fetlock OC, 5 QTL for fetlock OCD, and 3 QTL for hock OC. For POFs, we could determine 6 QTL. On ECA4q, a QTL for POFs in fetlock joints was identified at a relative position of 70.0–73.3 cM between the markers COR089 and HTG009 (Wittwer, Löhring, et al. 2007). Using a subset of 4 half-sib families, a QTL for fetlock OCD was detected in the same chromosomal region. The aim of this study was to confirm this QTL for fetlock OCD using single nucleotide polymorphisms (SNPs) of candidate genes in this genomic region and then to test these intragenic SNPs for association with fetlock OCD.
The recently improved human–equine comparative map for horse chromosome 4q12–q22 allows the comparisons of locations of genes implicated in osteoarthritis in humans for the identified QTL region on ECA4q (Dierks et al. 2006). Further helpful tools in this respect are the 13 964 expressed sequence tags (ESTs) from a normalized equine articular cartilage cDNA library containing parts of the mRNA of different equine cartilage clones, the equine genomic bacterial artificial chromosome (BAC) end sequences, and the large number of equine whole–genome shotgun (WGS) sequences in the National Center for Biotechnology Information (NCBI) nucleotide database (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&; DB=nucleotide).
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Pedigree Structure and Sampling
For confirmation of the QTL region on ECA4q, 4 paternal half-sib families were included in the linkage analysis. Each of these families showed significant logarithm of the likelihood ratio (LOD) scores for the QTL for fetlock POFs and fetlock OCD on ECA4q. These 4 families consisted of 84 horses including 45 progeny, their 35 dams, and 4 sires.
Informative SNPs were identified using sequence information from the sires of these 4 half-sib families and further 4 unrelated sires. All SNPs developed for the QTL region on ECA4q were further evaluated in an association analysis. Here we included 32 affected and 64 unaffected horses with an age at radiological examination between 12 and 32 months. These horses are progeny of 8 sires and belong to the 4 half-sib families in which significant linkage to fetlock OCD was detected and to further 4 paternal half-sib families showing no linkage to POFs or signs of OC or OCD and whose sires were used to identify informative SNPs in the candidate genes investigated here. Osseous fragments located at the dorsal aspect of the sagittal ridge of the third metacarpal/metatarsal bones were considered as signs of OCD, and these horses were treated as affected by fetlock OCD. Animals without any signs of radiographic changes of OC at this site were classified as free from OC and OCD. Radiographic findings classified as isolated radiopaque areas palmarly/plantarly at the attachment sites of the short sesamoidean ligaments to the proximal phalanx were defined as POFs.
The horses included in this study were randomly sampled and radiographed at a mean age of 14 months. Of each horse, digital radiographs were taken from each fetlock and hock joint.
Identification of SNPs
The human–equine comparative map was used to choose 7 functional and positional candidate genes for OC in the identified QTL region on ECA4q. Equine polymerase chain reaction (PCR) primers for SNP identification were selected using sequences of ESTs comparatively mapped on equine chromosome 4q12–q22 (Dierks et al. 2006). The primer pairs in the carboxypeptidase, vitellogenic-like (CPVL), in the BMP-binding endothelial regulator (BMPER), in the anillin-, actin-binding protein (ANLN), in the engulfment and cell motility 1 (ELMO1), and in the acyloxyacyl hydrolase (AOAH) gene with exception of the 3'-untranslated region (UTR) were designed using equine WGS and the Primer3 program (http://frodo.wi.mit.edu/) after masking repetitive elements with the RepeatMasker (http://www.repeatmasker.org/). For ANLN and ELMO1 3 primer pairs, respectively, were developed and the amplicons sequenced for 8 sires, but it was not possible to detect any DNA polymorphisms. The PCRs were performed in a total volume of 30 µl containing 10 ng of genomic DNA as template, 10 pmol of each primer, and 1 U Taq polymerase (MP Biomedicals, Eschwege, Germany). After a 4-min initial denaturation at 94 °C, 37 cycles of 45 s at 94 °C, 60 s at 60 °C, and 80 s at 72 °C were performed in MJ Research thermocyclers (Biozym, Hessisch Oldendorf, Germany). All these PCR products were sequenced on a MegaBACE 1000 (GE Healthcare, Freiburg, Germany) automated capillary sequencer, and the resulting sequences were searched for SNPs by visual inspection using the Sequencher 4.7 program (GeneCodes, Ann Arbor, MI). The sequencing reactions were carried out using the DYEnamic ET Terminator Cycle Sequencing Kit (Amersham Biosciences, Freiburg, Germany). Amplification started with an initial denaturation at 94 °C for 1.5 min, followed by 34 cycles of 20 s denaturing at 94 °C, 15 s annealing at 50 °C, and 2-min elongation at 60 °C. Finally, the reaction was cooled down to 4 °C for 10 min. The reaction product was cleaned up using the Sephadex G50 filtration kit (GE Healthcare, Freiburg, Germany).
Data Analysis
Multipoint nonparametric linkage analysis was performed using Merlin, version 1.0.1 (multipoint engine for rapid likelihood inference, Center for Statistical Genetics, University of Michigan, MI, Abecasis et al. 2002). The Zmean and LOD score test statistics were used to test for the proportion of alleles shared by affected individuals identical by descent for the considered marker loci (Kong and Cox 1997). Linkage analysis was performed for 4 half-sib families including 84 horses.
Correctness of Mendelian inheritance of markers was confirmed using PEDSTATS software (Wigginton and Abecasis 2005). The ALLELE procedure of the software package SAS/Genetics (Statistical Analysis System, Version 9.1.3, SAS Institute Inc., Cary, NC, 2007) was used to estimate the mean observed heterozygosity (Ho), the polymorphism information content and to test for Hardy–Weinberg equilibrium (HWE) of SNPs genotyped in the 96 horses. HWE was tested comparing observed and expected genotypic or allelic frequencies based on 
2-statistics. Markers not in HWE were regenotyped to preclude genotyping errors causing deviations from HWE. Overestimation of genotypic or allelic associations may be expected when markers are not in HWE. Preliminary association analyses were performed using the CASECONTROL procedure of SAS version 9.1.3, in order to test for disequilibrium between the phenotypes for OC or POFs and marker genotypes as well as marker alleles and number of alleles (trend in alleles). In order to control for systematic environmental and quantitative genetic effects in addition to the genotypic effects of the SNP markers showing significant effects in the 2-tests of the CASECONTROL procedure, we employed an animal model for the association analysis between the genotypes of the 6 AOAH SNPs and fetlock OCD. As all other association tests between the SNP markers and the different phenotypes for OC and POFs were not significant, animal model analyses were confined to fetlock OCD and SNPs within the AOAH gene. The linear animal model included the fixed effects of AOAH SNP genotype (Genotype_AOAH), sex, month of birth (Month), age at radiological examination of the horse (Age), interaction between age at radiological examination and sex, interaction between month of birth and sex, and random additive genetic effects of the animal (al).
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The additive genetic effects of the AOAH-associated SNP markers were estimated by pairwise comparison of the least square means of the 2 homozygous genotypes, and the dominance effects were calculated as the deviation of the least square means of the heterozygotes from the average of the 2 homozygous genotypes. Significance was tested using F-tests. The analyses were performed using PEST (Groeneveld 1990). Multiple testing was regarded using Bonferroni correction.
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Results and Discussion |
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TopMaterials and MethodsResults and DiscussionSupplementary MaterialFundingReferences |
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A total of 22 SNPs and 1 microsatellite marker were found in 6 candidate genes in the region from 70.0 to 73.3 cM on ECA4q14–q21.3 using 8 unrelated SGC stallions (Supplementary Table 1). The progeny of 4 stallions being heterozygous for the respective SNP was genotyped and used for linkage analysis to refine the putative QTL in SGC horses. After including 22 SNPs in the linkage study, located in 5 genes around the putative QTL, the SNP in the 3'-UTR of the AOAH gene (AJ543065:g.703A>G) affirmed the linkage for fetlock OCD at 72.2 cM with a Zmean of 2.04 (P = 0.03) (Figure 1).
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The SNP AM072940:g.230T>C in the PTHB1 gene was not in HWE and therefore was not used for association tests in order to avoid overestimation of genetic effects. Analyses for associations with POFs revealed no significant results (data not shown). Three SNPs in the AOAH gene at a relative position of 72.2 cM on ECA4q (ti|1248907742:g.288G>A, ti|1248907742:g.578C>T, AJ543065:g.703A>G) that were located in intron 8, intron 9, and the 3'-UTR in exon 21 (Figure 2) were significantly associated (P = 0.022–0.033) with fetlock OCD (Table 1). All SNPs in the AOAH gene were therefore tested in a more refined statistical model (Tables 2–4). Here, the SNP in the 3'-UTR (AJ543065:g.703A>G) showed an additive genetic effect for fetlock OCD of 0.42 ± 0.13 (P = 0.002) and a significant dominance effect of –0.32 ± 0.15 (P = 0.034). All homozygous G/G horses were affected and unaffected horses were predominantly homozygously A/A or heterozygously A/G (Tables 3 and 4). Contrasts of genotypic values for the risk to be affected by fetlock OCD between the genotype G/G and the other 2 genotypes were significant (P < 0.01).
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) and their SEs between genotypes of the intragenic AOAH SNP (AJ543065:g.703A>G) using MMSs of the risk for developing fetlock OCD in 96 SGC horsesOur study presents an association between a SNP (AJ543065:g.703A>G) in the AOAH gene and fetlock OCD which could serve as a suitable marker for fetlock OCD in SGC horses. The AOAH is a 2-subunit lipase that selectively hydrolyzes the secondary fatty acyl chains from the lipid A region of bacterial endotoxins. Further functions of this gene have not yet been determined. Therefore, the role of AOAH in the development of OC or bone morphogenesis is not clear. The human PTHB1 gene that is located very closely to the microsatellite marker HTG007 might be a functional candidate gene for OC. However, the intragenic SNPs developed for PTHB1 were not in linkage disequilibrium with signs of OC or POFs in our study. PTHB1 is downregulated by parathyroid hormone (PTH) in osteoblastic cells, and therefore, is thought to be involved in PTH action in bones (Adams et al. 1999). Another functional candidate gene BMPER gene was shown to be involved in BMP2- and BMP4-dependent osteoblast differentiation and BMP-dependent differentiation of the chondrogenic cells (Binnerts et al. 2004). At present, we could not identify SNPs in this gene, which were associated with OCD or POFs in SGC horses. Mutation analysis of AOAH and expression analyses will be helpful to clarify whether AOAH plays a role in the aetiopathogenesis of the OC syndrome in horses.
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Supplementary Material |
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Supplementary material can be found at http://www.jhered.oxfordjournals.org/.
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Funding |
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German Research Council, DFG, Bonn (DI 333/12-2); the Bavarian State Ministry for Food and Agriculture, Munich (A/04/16).
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Corresponding Editor: Ernest Bailey
Received December 8, 2006
Accepted October 26, 2007
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