Journal of Heredity Advance Access originally published online on March 29, 2007
Journal of Heredity 2007 98(3):267-271; doi:10.1093/jhered/esm004
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Brief Communications |
Molecular Genetic Analysis of the ATP2A2 Gene as Candidate for Chronic Pastern Dermatitis in German Draft Horses
From the Institute for Animal Breeding and Genetics, University of Veterinary Medicine Hannover, Foundation, Bünteweg 17p, 30559 Hannover, Germany
Address correspondence to O. Distl at the address above, or e-mail: ottmar.distl{at}tiho-hannover.de.
Chronic pastern dermatitis predominantly affects draft horses, and this condition is characterized by hyperkeratotic–hyperplastic dermal alterations. Chronic pastern dermatitis resembles the acral-hemorrhagic phenotype of Darier-White disease in humans. The ATP2A2 gene has been shown to be responsible for human Darier-White. Thus, we chose ATP2A2 on equine chromosome 8 (ECA8) as candidate for chronic pastern dermatitis in coldblood horses. A linkage analysis was performed in 10 paternal half-sib families consisting of 85 German coldblood horses using a microsatellite closely linked to ATP2A2, a microsatellite within intron 5, 3 single-nucleotide polymorphisms within intron 3 of ATP2A2, and 5 more distantly located flanking microsatellites on ECA8. These markers were clearly not linked to pastern dermatitis and so our data proved that ATP2A2 is not responsible for chronic pastern dermatitis of German coldblood horses.
The mammalian ATPase, Ca++ transporting, cardiac muscle, slow twitch 2 (ATP2A2) gene encodes the SERCA2 isoform of the Ca2+ pump, which belongs to the family of P-type cation pumps. SERCA2 is an enzyme, which catalyzes the ATP hydrolysis coupled with the translocation of calcium from the cytosol to the sarcoplasmic reticulum lumen, and is involved in regulation of the contraction–relaxation cycle. Mutations in ATP2A2 were previously shown to be responsible for the Darier-White disease in humans (Ruiz-Perez et al. 1999; Sakuntabhai et al. 1999). In the meantime, more than 100 different DNA mutations in ATP2A2 associated with the Darier-White disease were found, which were in most cases missense mutations. An acral-hemorrhagic type of this disease is known, in which hemorrhage into acantholytic vesicles on the palms and fingers cause black macules and incrustations (Ruiz-Perez et al. 1999). Similar clinical signs are found in chronic pastern dermatitis of draft horses. In horses affected by this disease, the skin of the palmar and plantar aspects of the fetlocks up to the carpal and tarsal joints shows plaque-like lesions, tuberous skin masses, or verruccous lesions (Wallraf et al. 2004a). Hyperkeratosis and epidermal hyperplasia with markedly increased numbers of Ki-67–positive basal epidermal cells were the main histopathological findings in German coldblood horses (Geburek et al. 2005). In more severe cases, the epidermal hyperplastic changes were more pronounced which indicated an abnormal differentiation of keratinocytes (Geburek et al. 2005). Chronic pastern dermatitis is most commonly seen in coldblood horses and particularly in the Belgium and Rhenish German coldblood. A large random sample of German coldblood horses including 909 horses from all different German coldblood breeds was examined for chronic pastern dermatitis in 2001 and 2002 (Wallraf et al. 2004b). The mean age of horses examined was 9.4 years with a range from 2.5 to 26 years. All 4 limbs were examined for skin alterations. Skin lesions were classified as erythema, exudation, crust formation, scaling, hyperkeratotic and hyperplastic plaque-like lesions, erosions, tuberous, or verrucosous masses with rugged surfaces. The prevalence of chronic pastern dermatitis in this sample of all German coldblood breeds was 66.4% with highest prevalences in Rhenish German (96.1%) and its locally derived strains, such as Saxon-Thuringa (84.3%) and Mecklenburg (76.4%), whereas in Black Forest and South German draft horses the prevalences were lower with values of 47.5% and 58.5% (Wallraf et al. 2004b). The influence of the breed was significant (P < 0.001). The most prevalent type of skin lesions in all breeds were crust formation (52.6%) and hyperkeratotic–hyperplastic skin alterations (47.7%). All 4 limbs or both hind limbs were affected most frequently. Whereas the Darier-White disease is known to be autosomal dominantly inherited, the mode of inheritance for chronic pastern dermatitis is not yet known. However, heritabilities of 0.14–0.20 have been estimated for the prevalence of chronic pastern dermatitis in an animal model analysis using records of 455 South German coldblood horses (Wallraf et al. 2004a). Thus, the ATP2A2 gene located on equine chromosome 8 (ECA8) was chosen as a candidate for a quantitative trait locus of chronic pastern dermatitis in coldblood horses. Subsequently, we used a gene-associated microsatellite marker and 3 gene-associated single-nucleotide polymorphisms (SNPs) derived from a shotgun BAC sequence (accession no. AM137440), and in addition, a highly polymorphic microsatellite marker flanking the ATP2A2 gene on ECA8 was employed for a multipoint linkage analysis in a half-sib design. Markers flanking ATP2A2 were used in order to disclose recombinations for the genomic region surrounding ATP2A2.
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Materials and Methods |
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Pedigree Structure and Sampling
For the linkage analysis, we collected blood samples from 85 horses of the breeds South German and Saxon-Thuringa. They belong to 10 paternal half-sib families segregating for horses showing chronic pastern dermatitis and nonaffected horses with at least 5 years of age. The families consisted of 5–13 individuals. The average paternal half-sib family size was 8.5 individuals. Among the progeny there were 78 females and 6 male horses. A blood sample of the common sire was available for one family. Dams could not be sampled due to the high age of horses examined. Because of the sometimes late onset of the disease, we preferentially included affected individuals in our analysis. The prevalence of chronic pastern dermatitis was 85.9% in the genotyped animals (Table 1). The mean age of horses at examination was 9.3 ± 2.7 years with a range from 5 to 15 years.
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Marker Analysis
Genomic DNA from the ethylenediaminetetraacetic acid blood samples was extracted by using the QIAamp 96 Spin Blood Kit (Qiagen, Hilden, Germany). For the scan of ECA8, we used a microsatellite marker located within intron 5 of the ATP2A2 gene (ATP2A2_MS5, Müller et al. 2006) and an already published microsatellite marker (LEX023, Coogle et al. 1996). In addition, we used 5 previously published microsatellite markers (COR003 and COR012, Hopman et al. 1999; COR056, Ruth et al. 1999; COR097, Tallmadge et al. 1999; UCDEQ046, Shiue et al. 1999) in order to determine the number of recombinants and nonrecombinants among the horses affected. The positions of the microsatellite markers referred to the linkage map of Swinburne et al. (2006). ATP2A2_MS5 is closely linked to the microsatellite LEX023 (Müller et al. 2006), and the distance of LEX023 to ATP2A2_MS5 is about 2 cM. Furthermore, we identified 3 SNPs in intron 3 of ATP2A2 (accession no. AM137440). We designed an intragenic primer pair using Primer3 that amplified a sequence that contained these 3 SNPs (AM137440 [GenBank] :g.2804C>T, AM137440 [GenBank] :g.2581T>A, and AM137440 [GenBank] :g.3000T>A) in a polymerase chain reaction (PCR) product. At least one of these 3 SNPs was informative for each one of the paternal half-sib families analyzed.
All PCRs were carried out in 12 µl reaction mixtures containing 2 µl genomic DNA (10 ng/µl), 1.2 µl 10x PCR buffer, 0.24 µl dimethyl sulfoxide, 0.5 µl of each primer (10 pmol/µl), 0.2 µl dNTPs (5 mM each). and 0.1 µl Taq polymerase (5 U/µl) (Qbiogene, Heidelberg, Germany). For amplification, PTC 100TM or PTC 200TM thermal cyclers (MJ Research, Watertown, MA) and a general PCR program with variable annealing temperature were used. The reaction started with denaturing all samples at 94 °C for 4 min followed by an empirically determined amount of cycles (32–35) comprising denaturation for 30 s at 94 °C, annealing for 30 s at 56–60 °C, and extension for 45 s at 72 °C. The PCR was completed with a final cooling at 4 °C for 10 min.
For the analysis of the microsatellite marker genotypes, all PCR products were size fractionated by gel electrophoresis on an automated sequencer (LI-COR 4300, Lincoln, NE) using 6% polyacrylamide denaturing gels (Rotiphorese Gel 40, Roth, Karlsruhe, Germany). Allele sizes were scored against IRD 700– and IRD 800–labeled DNA ladders used as standards on every gel. Alleles were assigned by visual examination.
The PCR products from all 85 horses used for SNP detection were directly sequenced with the DYEnamic ET Terminator kit (Amersham Biosciences, Freiburg, Germany) and a MegaBACE 1000 capillary sequencer (Amersham Biosciences).
Nonparametric Linkage Analysis
A nonparametric multipoint linkage analysis was carried out using the polymorphic microsatellite marker LEX023, the gene-associated microsatellite ATP2A2_MS5, 3 intragenic SNPs for the ATP2A2 gene, and the 5 polymorphic microsatellites distributed over ECA8. The nonparametric multipoint linkage analysis was based on allele sharing by identical by descent (IBD) methods and the MERLIN 1.0.1 software was employed (Abecasis et al. 2002). Haplotypes were estimated using the option "best" of the MERLIN 1.0.1 software. Recombinants and nonrecombinants among horses affected were counted based on these haplotypes and used to calculate a log of odds (LOD) score according to Ott (1991). Nonparametric linkage analyses do not require assumptions on the mode of inheritance and the genetic parameters of the specified model and so this approach should be useful for traits under unknown inheritance models. Z-mean and LOD score were used to test the proportion of alleles shared by affected individuals that were IBD for the marker loci. For these families, the maximum (minimum) achievable Z-mean was 19.17 (–2.59), and the corresponding maximum (minimum) value for the LOD score was 7.99 (–0.29). Under the null hypothesis of no linkage between marker alleles and disease, half sibs would be expected to share half of their paternal alleles. In the case of no linkage and equal distribution of alleles among affected relatives, Z-mean approaches the minimum achievable value. When linkage is present under the alternative hypothesis, the proportion of paternal alleles IBD would be significantly deviate from the expected IBD proportions of the null hypothesis. We employed multipoint analyses in order to use marker information most efficiently and to increase power of the linkage tests.
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Results and Discussion |
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TopMaterials and MethodsResults and DiscussionReferences |
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Information Content of Markers Used
The highly polymorphic microsatellite marker in intron 5 of the ATP2A2 gene showed a (GT)25 repeat motif. This microsatellite marker showed 11 alleles, a heterozygosity of 58.3%, and a polymorphism information content (PIC) of 58.0% in the horses genotyped. Furthermore, the microsatellite marker LEX023 had 7 alleles, a mean PIC of 73.4%, and a mean heterozygosity of 82.5%. Using the 2 microsatellites, the mean PIC per family was between 41.0% and 74.6%, indicating that all half-sib families were informative for these markers (Table 1).
Linkage Analysis
The nonparametric multipoint linkage analysis in 10 paternal half-sib groups did not show significant test statistics. The Z-means for LEX023, ATP2A2_MS5, and the 3 intragenic SNPs were between –0.68 and –0.71; the LOD scores were between –0.07 and –0.08; whereas the error probabilities ranged from 0.7 to 0.8 (Table 2). Heterogeneity of Z-means and LOD scores between paternal progeny groups could not be shown (Table 3). Obviously, no paternal haplotype was associated with chronic pastern dermatitis in these coldblood horses (Figure 1). The paternal haplotypes were distributed among affected progeny closely to the expected proportion of 50%, and thus, no excess of a certain haplotype could be observed in the affected horses. Recombinants between the intragenic and flanking markers could be observed in 34 horses affected, and the number of nonrecombinants was 24 among horses affected. The LOD score based on these figures was –12.25 for the average distance between intragenic markers and recombination events (r = 0.15). Linkage is clearly rejected when this large number of recombinants (59%) identified via their haplotypes is considered. ATP2A2 is eliminated as a causative gene being involved in the pathogenesis of chronic pastern dermatitis in South German and Saxon-Thuringa coldblood horses because there was no evidence for linkage of intragenic markers. However, the ATP2A2 gene–associated marker may serve for further linkage studies in horses.
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Acknowledgments |
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We wish to thank Prof Dr E. Deegen and Dr B. Ohnesorge, Clinic for Horses of the University of Veterinary Medicine Hannover, Foundation, for their expert assistance in clinical examinations. We appreciate the assistance of Dr A. Wallraf and Dr P.G. Meyer in sampling horses for this study.
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Footnotes |
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Corresponding Editor: Ernest Bailey
Received January 3, 2006
Accepted December 27, 2006
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References |
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