General discussion
169
General discussion
A whole genome scan for the identification of the chromosomal locations of quantitative trait loci (QTL) for equine osteochondrosis (OC) and osteochondrosis dissecans (OCD) was performed in order to identify positional candidate genes. The high number of QTL on different chromosomes found for OC and OCD in this study suggests that several genes are possibly involved in the development of OC and OCD. However we cannot conclude on the type of gene action and in which way the different genes may interact with each other. So it is not possible to decide if OC already develops if at least one or both alleles of one responsive gene are mutated or only in the case, when mutations at several gene loci are present. The QTL for OC in fetlock and hock joints found in the present analysis were for the most part heterogeneously distributed. The QTL on ECA2 at 25.00-56.00 cM detected for fetlock OC and OCD was also found for hock OC. ECA16 is harbouring QTL at 0.00-49.00 cM for fetlock OC and OCD as well as for hock OC and OCD. An explanation for this might be the overlap of animals which are affected by fetlock and hock OC simultaneously. All other QTL for the traits fetlock and hock OC were mapped to different chromosomes. This may indicate that the genetic influences on the development of fetlock OC and hock OC are mostly due to different loci. This seems likely as the genetic correlations between fetlock OC and hock OC were close to zero in trotter horses (Grøndahl and Dolvik 1993), or even negative in Hanoverian warmblood horses (Stock et al. 2005). The QTL on ECA2 at 26.91-41.70 cM for the trait OC fetlock reached also genome-wide significance.
Candidate genes in the QTL flanking regions may be chosen by means of comparative human-equine maps. Potential candidate genes have to code for hormones, enzymes, metabolic factors and/or their receptors, which are involved in the complex of cartilage maturation and differentiation during enchondral ossification or in growth processes. Candidate genes may be also involved in osteoarthritis of other species. Furthermore, the Equine Articular Cartilage cDNA Library may be helpfully to select candidate genes. At the moment, 13,964 equine articular EST’s can be found in the NCBI nucleotide database (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=nucleotide).
Andersson-Eklund et al. (2000) identified three QTL for OC on pig chromosomes SSC5, 13 and 15. Possible candidate genes derived from QTL positions might be pituitary specific transcription factor 1 (PIT1), which codes for a transcriptional factor of growth hormone and one gene coding for parathyroid hormone receptors (PTHR) for the chromosome-wide QTL on SSC13, and insulin-like growth factor I (IGF-I) and cartilage homeoprotein1 (CART1) for the genome-wide QTL on SSC5, respectively.
These genes were selected on the one hand for their indicated role in the development of OC or cartilage growth and on the other hand due to their locations in the vicinity of an identified QTL or in the homologous region of the human genome. A putative correspondence was identified between the QTL on SSC13 and the QTL on ECA16 observed in the present study. Lee et al. (2003) also detected QTL associated with OC related traits in pigs on SSC7 and SSC16 but none of these QTL exceeded the chromosome-wide significance level.
Due to the high number of QTL found for OC and OCD, two QTL located on ECA2p and ECA4q were chosen for further refinement. A high-resolution comparative map is important for the fine mapping of QTL and for finding positional candidate genes that might influence an investigated trait. Consequently, the genomic region, which has to be searched for candidate genes, can be identified with higher accuracy by increasing the marker density of the map used. It was difficult to locate positional candidate genes for the QTL region on ECA4 by using the existing equine-human comparative maps (Chowdhary et al. 2003, Milenkovic et al. 2002, Swinburne et al.
2006), because the syntenic human regions could not be identified or resolved with the necessary accuracy.
The COL8A2 (collagen, type VIII, alpha 2), COL16A1 (collagen, type XVI, alpha 1), COL9A2 (collagen, type IX, alpha 2) and MATN1 (matrilin 1, cartilage Matrix Protein) genes were chosen to be mapped on ECA2. The RH and FISH mapping results of the equine COL16A1 gene on ECA2p15.1-p15.3, the COL8A2 gene on ECA2p15-p16, COL9A2 gene on ECA2p15-p16 and the RH mapping results of the equine MATN1 gene agree with comparative mapping of the current equine-human comparative maps (Chowdhary et al. 2003, Milenkovic et al. 2002, Swinburne et al.
2006) of ECA2p, which shows conserved synteny to HSA1p. The structure of one promising functional candidate gene, the COL9A2 gene was analyzed. The equine COL9A2 gene consists of 32 exons in agreement with the human and murine
General discussion
171
COL9A2 genes. The total size of the equine COL9A2 gene is approximately 15 kb.
The mRNA of the equine COL9A2 gene contains an open reading frame of 2,067 nt coding for a protein of 688 amino acids. The search for sequence variations within the COL9A2 gene revealed a total of 32 SNPs. One SNP located in exon 18 affects the amino acid sequence at position 310 of the equine COL9A2 protein. This SNP causes an amino acid exchange from threonine to alanine. Quality evaluation of the observed SNPs revealed that eleven SNPs were suitable for linkage studies in horses.
A high-resolution comparative map of ECA4q12-q22 with an improved breakpoint resolution was constructed, because the previously published RH map only included two type I markers for ECA4q12-q22 (Chowdhary et al., 2003). In comparison to the current RH map (Chowdhary et al., 2003), differences were found in marker order as well as marker arrangements. The comparative map of ECA4 and HSA7 presented here should lead to improvement of QTL studies and the selection of candidate genes affecting OC for which QTL have been mapped on this equine chromosome.
Localisation of QTL for OC needs the refinement of its position through single nucleotide polymorphisms (SNPs) as known equine mirosatellites are too sparsely distributed on the equine maps. Development of SNPs is preferentially performed using BAC end sequences and equine ESTs corresponding to candidate genes associated with human osteoarthritis, and further equine ESTs located in the QTL regions. The identification of intragenic SNP markers includes the possibility of finding mutations that cause the phenotypic effects. However, it is sometimes difficult to prove, that a certain mutation is in fact the causative trait influencing mutation and not just another one in very close linkage to it. A total of 16 SNPs were identified within equine BAC end and EST sequences on ECA2 and used for linkage analysis.
Additionally, three SNPs in the COL9A2 gene were used for linkage analysis. The significant Zmean-values on ECA2 found by Böneker et al. (2006) were confirmed by adding the SNP markers and the QTL could be narrowed down by haplotype analysis to an interval from 29.54 to 31.4 cM. This region includes a gene which is involved in the development of OC in fetlock joints in horses. One promising candidate, the MATN1 gene is located at approximately 30 cM on ECA2. However, more SNPs in this region have to be developed and genotyped in informative families, in order to find the responsible gene. For ECA4 a total of 15 SNPs were identified within equine
BAC end and EST sequences and used for linkage analysis. One SNP in the parathyroid hormone-responsive B1 (B1) gene of ECA4 was significantly associated with hock OC, whereas all other SNPs of ECA4 showed neither significant linkage disequilibrium nor association with OC. An association with osteochondrosis in fetlock joints was unlikely, so one might conclude that possibly the B1 gene is only involved in the pathogenesis of hock OC. This QTL region should be scanned for further SNPs associated with hock OC. With the help of associated SNPs and mRNA analyses it should be possible to unravel the responsible genes.
The precondition for evaluating responsible candidate genes for OC is to improve the equine maps by increasing the marker density. For all regions harbouring quantitative trait loci (QTL) for OC comparative equine-human maps have to be constructed.
Then single nucleotide polymorphisms (SNPs) should be developed for all positional candidate genes to perform linkage and association analyses. Using linkage analyses the LOD scores might decrease due to the fact that the mutation is of importance only in a few families. In that case, the families showing highest values for LOD score can be chosen for the further refinement of QTL. Once a genomic QTL region for OC has been successfully narrowed down to 1-5 Mb, further positional candidate genes from the conserved chromosomal region in man can be identified for testing linkage disequilibrium and association with OC by genotyping SNPs for more animals with known OC phenotype. If a population-wide linkage disequilibrium or significant association for SNPs with OC was found, the causal mutations may be discovered by sequencing of at least all the exons with their splice sites and the 5’- and 3’- untranslated regions of the candidate genes of a large sample with affected and non-affected horses from different families.
When a putative mutation has been identified it is necessary to provide evidence that the mutation causes the phenotypic effect. Differential cDNA expression analysis of the cartilage or synovial fluid of affected and non-affected horses by means of human high-density-cDNA-microarrays may help to elucidate the causal gene. Semevolos et al. (2005) determined changes in the expression of different genes in horses affected with OC by using cartilage, harvested from OC affected joints and real-time quantitative polymerase chain reaction. The present study was able to identify QTL regions in the horse genome and a marker for osteochondrosis in hock joints which is already suitable in horse breeding. This is the first study showing significant QTL for equine osteochondrosis.
General discussion
173 References
Andersson-Eklund L, Uhlhorn H, Lundeheim N, Dalin G, Andersson L (2000) Mapping quantitative trait loci for principal components of bone measurements and osteochondrosis scores in a wild boar x large white intercross. Genet Res 75, 223-230
Böneker C, Löhring K, Wittwer C, Drögemüller C, Distl O (2006) Genome-wide search for markers associated with osteochondrosis in Hanoverian Warmblood horses. submitted
Chowdhary BP, Raudsepp T, Kata SR, Goh G, Millon LV, Allan V, Piumi F, Guerin G, Swinburne J, Binns M, Lear TL, Mickelson J, Murray J, Antczak DF, Womack JE, Skow LC (2003) The first-generation whole-genome radiation hybrid map in the horse identifies conserved segments in human and mouse genomes.
Genome Res 13, 742-751
Grøndahl AM, Dolvik NI (1993) Heritability estimations of osteochondrosis in the tibiotarsal joint and of bony fragments in the palmar/plantar portion of the metacarpo- and metatarsophalangeal joints of horses. JAVMA 203, 101-104 Lee GJ, Archibald AL, Garth GB, Law AS, Nicholsen D, Barr A, Haley CS (2003)
Detection of quantitative trait loci for locomotion and osteochondrosis-related traits in Large White X Meishan pigs. Animal Science 76, 155-165
Milenkovic D, Oustry-Vaiman A, Lear TL, Billault A, Mariat D, Piumi F, Schibler L, Cribiu E, Guerin G (2002) Cytogenetic localization of 136 genes in the horse:
comparative mapping with the human genome. Mamm Genome 13, 524-534 Semevolos SA, Strassheim ML, Haupt JL, Nixon AJ (2005) Expression patterns of
hedgehog signaling peptides in naturally acquired equine osteochondrosis. J Orthop Res 23, 1152-1159
Stock KF, Hamann H, Distl O (2005) Estimation of genetic parameters for the prevalence of osseous fragments in limb joints of Hanoverian Warmblood horses. J Anim Breed Genet 122, 271-280
Swinburne JE, Boursnell M, Hill G, Pettitt L, Allen T, Chowdhary B, Hasegawa T, Kurosawa M, Leeb T, Mashima S, Mickelson JR, Raudsepp T, Tozaki T, Binns M (2006) Single linkage group per chromosome genetic linkage map for the horse, based on two three-generation, full-sibling, crossbred horse reference families. Genomics 87, 1-29
Chapter 11
Summary
Summary
177
Summary
Claudia Dierks geb. Böneker (2006)
Molecular genetic analysis of quantitative trait loci (QTL) for osteochondrosis in Hanoverian warmblood horses
The aim of this work was to perform a whole genome scan for the identification of quantitative trait loci (QTL) for equine osteochondrosis (OC), to identify positional candidate genes for this disease and to develop marker tests based on intragenic single nucleotide polymorphisms (SNPs).
The first step of the whole genome scan included 157 microsatellite markers, genotyped for 104 progeny of 14 paternal half-sib families of Hanoverian warmblood horses, their dams and eight sires. The marker genotypes, the pedigree data and the phenotype data of the foals were analyzed using non-parametric linkage analysis based on identical by descent (IBD) mapping. Traits used were OC (fetlock and/or hock joints affected), OCD (fetlock and/or hock joints affected), fetlock OC, fetlock OCD, hock OC and hock OCD. In a second step 61 additional markers were chosen to refine putative QTL found in the first scan. Nineteen chromosome-wide significant QTL with error probabilities for LOD scores or Zmeans ≤ 0.05 for OC and OCD were located on 17 different equine chromosomes: 2, 3, 4, 5, 7, 8, 9, 13, 14, 15, 16, 18, 19, 21, 22, 24 and 30. QTL for fetlock OC and hock OC were mostly mapped on different chromosomes, indicating that these traits may be inherited independently. A genome-wide QTL was found on ECA2p for fetlock OC and fetlock OCD. Due to the high number of QTL found for OC and OCD, the genome-wide QTL located on ECA2p and a chromosome-wide QTL located on ECA4q were chosen for further refinement.
Three positional candidate genes located on ECA2p (COL8A2, COL16A1 and COL9A2) were mapped in horses for the first time by fluorescence in situ hybridization (FISH) and radiation hybrid (RH)-mapping. The complete genomic sequence of the equine collagen, type IX, alpha 2 (COL9A2) gene was determined.
Thirty-two SNPs in COL9A2 were detected in a mutation scan of eight unrelated
Hanoverian warmblood stallions, including a SNP in Exon 18 that affects the amino acid sequence of COL9A2. A comprehensive 5,000-rad radiation hybrid (RH) map of a 40 cM region on equine chromosome 4q12-q22 harbouring quantitative trait loci for equine OC was constructed including 29 gene-associated sequence tagged site (STS) markers. Three blocks of conserved synteny between horse and man were identified showing two chromosomal breakpoints in the segment of ECA4q12-q22.
Breakpoint resolution in the human-equine comparative map was considerably improved.
Furthermore, 31 new SNP-markers located on ECA2p and ECA4q were developed.
Therefore, PCR primers were designed on the basis of BAC end and equine EST sequences. The PCR products of eight unrelated stallions were screened for SNPs.
All newly developed 31 SNPs and three SNPs in the COL9A2 gene were used for fine mapping of the QTL locations for OC on ECA2p and ECA4q identified by the whole genome scan. The inclusion of SNP markers improved the test statistics on ECA2 as compared to the whole genome scan where only microsatellites were used.
Regarding ECA2, the Zmean was highest in the interval from 29.51 to 30.09 cM. The functional candidate gene COL9A2 is located in the proximity of this interval. For ECA4q the highest Zmean-value was reached at 66 cM. A SNP in the B1 gene of ECA4 was significantly associated with hock OC and hock OCD, whereas all other SNPs on ECA4 did not show significant linkage disequilibrium or association with traits of OC. The additive genetic effects of this B1 SNP were -0.85 for hock OC and -0.83 for hock OCD and the significant dominance effects for hock OC were 0.47 and 0.53 for hock OCD.
These results indicate that genes which are involved in the development of OC are located on ECA2p and ECA4q. The refinement of the two QTL found for OC was a first step towards the identification of genes responsible for equine OC. An intragenic SNP marker of the B1 gene on ECA4q showed a population-wide linkage disequilibrium for the traits hock OC and OCD, and appeared as a suitable marker for these traits in Hanoverian warmblood horses.
Chapter 12
Zusammenfassung
Zusammenfassung
181