Article

Osteochondrosis (OC) is an inherited developmental orthopaedic disorder in young horses characterized by abnormal chondrocyte differentiation and maturation (Jeffcott & Henson 1998). Articulations most commonly affected in horses are fetlock, hock and stifle joints. Whole genome scans in Hanoverian warmblood and South German Coldblood discovered quantitative trait loci (QTL) for osteochondrosis in fetlock and hock joints (Dierks et al. 2007; Wittwer et al. 2007). In Hanoverian warmblood horses chromosome-wide significant QTL were identified for OC and OCD in fetlock joints on horse chromosome 5 (ECA5) at 65.5-100.1 cM. The aim of this study was to refine the position of this QTL for fetlock OC and fetlock OCD on ECA5 using newly developed microsatellite markers.

We used 14 paternal half-sib families including a total of 211 horses for genotyping.

These horses were identical with the families used in the previous QTL study (Table S1). Diagnosis of osteochondrosis was done following the recording and evaluation

scheme developed for warmblood horses (Dierks et al. 2007). Sagittal ridge of the 3rd metacarpal/metatarsal bone of fetlock joints and intermediate ridge of the distal tibia, lateral trochlea of talus of hock joints and the medial malleolus of the tibia were considered as predilection sites for OC. Signs consistent with osteochondrosis were irregular bone trabeculation with variable radiolucency, irregular bone margin, new bone formation or osseous fragments only in the case when these changes were located at these predilection sites. Horses showing radiographic changes of osteochondrosis with or without osseous fragments at the predilection sites were classified as affected by fetlock OC and/or hock OC and those horses exhibiting radiodense bodies as signs for joint mice at the above mentioned predilection sites were treated as affected by osteochondrosis dissecans (OCD). Horses with pathological changes in fetlock or hock joints other than osteochondrosis were not used in our study. Animals without any signs of radiographic changes at all joints examined (fetlock, hock and stifle) were considered as free from OC and only these horses were included as controls.

For the refinement of the QTL for fetlock OCD on ECA5, 29 new microsatellites (ABGe010–ABGe031, ABGe135-ABGe141) were developed (Table S2). Genotyping was done according to Dierks et al. (2007).

Multipoint non-parametric linkage analysis (NPL) was performed using the Merlin software (multipoint engine for rapid likelihood inference, version 1.1.2) (Abecasis et al. 2002) for a total of 49 microsatellites (Table S2). The Zmean and LOD score test statistics were used to test for the proportion of alleles shared by affected individuals identical by descent (IBD) for the considered marker loci (Kong & Cox 1997;

Whittemore & Halpern 1994). Chromosome-wide significant linkage was determined using a permutation approach as described by Dierks et al. (2007). The maximum (minimum) achievable Zmeans were 9.07 2.78) for OC in fetlock joints, and 4.12 (-2.32) for OCD in fetlock joints. The corresponding maximum (minimum) values for LOD scores were 6.30 (-0.59) and 3.05 (-1.00) indicating enough power to detect genome-wide significant linkage. Genome-wide probabilities were obtained by applying a Bonferroni correction: Pgenome-wide = 1 – (1 – Pchromosome-wide)^1/r, where r = length of ECA5 (99.7 Mb) divided by the total equine genome length (2680 Mb).

In this study, two different analyses were performed for the following phenotypes: (1) OC present in fetlock joints or absent in these joints of all limbs, and (2) OCD present in fetlock joints or absent in these joints of all limbs.

In addition, the genotypic data was evaluated using the ALLELE and CASECONTROL procedures of SAS/Genetics (Statistical Analysis System, Version 9.2, SAS Institute, Cary, NC, USA 2008) to determine the observed heterozygosity (HET), the polymorphism information content (PIC) and Hardy-Weinberg equilibrium and to evaluate genotypic and allelic associations and the trend of the alleles with fetlock OC and fetlock OCD using χ²-tests.

The marker positions delimiting the QTL for fetlock OC and fetlock OCD from the previous whole genome scan on ECA5 (Dierks et al. 2007) were adjusted to 78.29-98.39 Mb on EquCab2 using BLAST analyses for the microsatellite flanking sequences on ECA15. The non-parametric multipoint linkage analysis showed chromosome-wide significant Zmeans and LOD scores in the region from 76.69 to 92.77 Mb on ECA5 for fetlock OCD and in the region from 79.65 to 89.31 Mb for fetlock OC (Fig. 1).

The highest Zmeans were 3.30 for fetlock OCD with corresponding genome-wide error probabilities of 0.01 at 79.65 to 83.74 Mb and at 86.56 to 89.31 Mb. The highest LOD score for fetlock OCD was 2.41 with a corresponding genome-wide error probability of 0.01 at the microsatellite ABGe138 at 78.03 Mb.

For the trait fetlock OC, chromosome-wide significant error probabilities were reached for both test statistics at 79.65 to 83.74 Mb and at 85.21 to 89.31 Mb. While the highest Zmean was 2.44 at 85.21 Mb with a chromosome-wide error probability of 0.007, the highest LOD score was 1.59 at 79.65 Mb with a chromosome-wide error probability of 0.003 (Table S3).

Significant genotypic and/or allelic association were found for fetlock OCD at 84.44 Mb (TKY525), 85.21 Mb (ABGe016), 86.56 Mb (ABGe018) and 91.52 Mb (ABGe025) (Table S4). This result also corroborates the QTL location in the region between 76.69 Mb and 92.77 Mb.

The approximate consistence of the QTL for fetlock OC and fetlock OCD supports to the assumption that the same genes may play a role in the development of this

disease and that OCD is an aggravated form of OC. The delimitation of the QTL using very dense microsatellite scans also allows clarifying how many positional candidate genes are located in the linked region. For that purpose we used the Equine Articular Cartilage cDNA Library (http://www.ncbi.nlm.nih.gov/sites/entrez) to search for genes which are expressed in equine cartilage and located within the QTL at 76.69 to 92.77 Mb on ECA5. From a total of 13,964 equine articular ESTs (expressed sequence tag), we identified 53 ESTs which correspond to 22 different genes (Table S5). Besides these cartilage expressed genes, collagen type XXIV, alpha 1 (COL24A1) was identified as a functional candidate gene at 78.1 Mb.

COL24A1 is a marker for embryonic bone formation and may play a role in regulation of type I collagen fibrillogenesis (Koch et al. 2003). Furthermore, Matsuo et al. (2008) found out that COL24A1 is not only expressed in the forming skeleton of the mouse embryo, but also transcribed in the trabecular bone and periosteum of the newborn mouse. Due to its function in bone formation COL24A1 seems to be a suitable functional candidate gene for osteochondrosis because this disease has its origin in the disturbance of ossification.