Türkiye'de üretilen hizmet köpeklerinde kalça displazisi ile ilişkili genetik markırların araştırılması
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Dosyalar
Tarih
2020
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Yayıncı
Trakya Üniversitesi Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/embargoedAccess
Özet
Gelişimsel ortopedik bir hastalık olan kalça displazisi, kalıtsal ve çevresel faktörlerin etkisi ile meydana gelir. Hem insanlarda hem de köpeklerde yaygın olarak görülmesine rağmen hastalığın genetik arka planı aydınlatılamamıştır. Köpek kalça displazisine (KKD) karşı, fenotipe dayanan seleksiyon programları uzun yıllarca uygulanmış olsa da istenilen başarı elde edilememiştir. Günümüzde, kalça displazisini erken teşhis edebilecek bir DNA testi geliştirmek için, hastalığa neden olan genetik lokuslar tespit edilmeye çalışılmaktadır. Bu araştırmada, KKD hastalığın gelişimi ile ilişkili olabilecek genetik varyasyonları tespit etmek için, bir vaka kontrol çalışması tasarlanmıştır. Türkiye’de hizmet köpeği olarak yetiştirilen, 53 adet Labrador Retriever (17 vaka, 36 kontrol), 63 adet Belçika Malinois (14 vaka, 49 kontrol) ve 56 adet Alman Çoban Köpeği (24 vaka ve 32 kontrol) araştırma kapsamına dahil edilmiştir. Moleküler genetik analizler yapılmak üzere, KKD hastalığının gelişimi ile ilişkili olabilecek 5 gen belirlenmiştir (CX3CR1, CHST3, OSTF1, IBSP, FGF12). Seçilen bu genlerden, CX3CR1 geninin 2. ekson ve 3' UTR bölgeleri DNA dizi analizi metodu ile araştırılarak varyasyonlar tespit edilmiştir. Diğer genler üzerinde ise, KKD gelişimi ile ilişkili olduğu, daha önce farklı araştırmacılar tarafından rapor edilmiş olan 5 adet TNP (CHST3 geni 5? UTR’de rs24076746 ve 3? UTR’de rs24105396, OSFT1 geni 4. intronda rs8995004, IBSP geni 3. intronda rs23759713 ve FGF12 geni 3. intronda rs851690466) RFLP metodu ile araştırılmıştır. Haplotiplerin belirlenmesi, miRNA bağlanma bölgelerinin tespit edilmesi ve TNP-TNP etkileşimlerinin KKD hastalığının gelişimi üzerine etkileri, biyoinformatik metotlar ile araştırılmıştır. Ki-kare değerleri, olabilirlik oranı (OR) ve %95 güven aralığı (GA) lojistik regresyon analizi ile hesaplanmıştır. CX3CR1 geninin 2. ekson bölgesinde varyasyon bulunamamıştır, 3' UTR bölgesinde ise 8 varyasyon (6 TNP, 1 insersiyon, 1 delesyon) tespit edilmiştir. Yapılan istatistiksel değerlendirmeler sonucunda, rs9022770 G>A ve rs9022769 G>A TNP’lerinin Belçika Malinois ırkı köpeklerde KKD gelişimi ile ilişkili (p<0,05) olduğu görülmüştür. Diğer iki köpek ırkında ise bireysel olarak varyasyonlar ile hastalık arasında istatistiki olarak anlamlı bir ilişki bulunamamıştır. Belçika Malinois ırkı köpeklerde, rs9022770 G>A TNP’si için A allelinin, p=0,002 anlamlılık düzeyinde patojen allel olduğu (OR=13,091, %95 GA=2,474-69,264) belirlenmiştir. rs9022769 G>A TNP’si için ise G allelinin p=0,011 anlamlılık düzeyinde patojen allel olduğu (OR=3,333, %95 GA=1,279-8,688) hesaplanmıştır. CX3CR1 rs9022769/rs852431030 TNP’lerinin oluşturduğu GT haplotipinin, p=0,022 anlamlılık düzeyinde patojen haplotip olduğu (OR=6,222, %95 GA=1,280-30,243) belirlenmiştir. Araştırılan diğer köpek ırklarında, oluşan haplotipler ile KKD gelişimi arasında istatistiksel olarak anlamlı bir ilişki tespit edilememiştir. Önceki araştırmacıların, KKD gelişimi ile ilişkili olduğunu rapor ettikleri 5 TNP’den sadece bir tanesi, IBSP geni 3. introndaki rs23759713 C>T TNP’si, Alman Çoban Köpeklerinde KKD gelişimi ile ilişkili bulunarak desteklenmiştir. rs23759713-T allelinin p=0,026 anlamlılık düzeyinde koruyucu allel olduğu (OR=0,207, %95 GA=0,048-0,891) belirlenmiştir. Diğer 4 TNP’nin (rs24076746, rs24105396, rs8995004, rs851690466) KKD gelişimi ile ilişkisi bulunamamıştır. Araştırmamız sonucunda, KKD gelişimi ile ilişkili olduğunu bulduğumuz CX3CR1 geninin 3' UTR polimorfizmlerinin (rs9022770, rs9022769), farklı köpek popülasyonlarında da çalışılıp doğrulandıktan sonra, KKD hastalığına karşı uygulanacak markör destekli seleksiyon (MAS) programlarına katkı sağlayacağını düşünmekteyiz. IBSP geni 3. intronundaki rs23759713 TNP’sinin ise KKD gelişimi ile ilişkisi çalışmamızda da desteklenmiştir. Araştırmamız, hem KKD hastalığın genetik arka planını aydınlatmak için, hem de MAS çalışmaları için faydalı olabilecek veriler sunmaktadır.
Hip dysplasia, a developmental orthopedic disease, occurs with the influence of hereditary and environmental factors. Although it is common in both humans and dogs, the genetic background of the disease has not been elucidated. Although the selection programs based on phenotype against canine hip dysplasia (CHD) have been applied for many years, the desired success has not been achieved. Today, to develop a DNA test that can diagnose hip dysplasia early, the disease causing genetic loci are being identified. In this study, a case-control study was designed to detect genetic variations that may be associated with the development of CHD disease. Breding as service dogs in Turkey, 53 Labrador Retrievers (17 case, 36 control), 63 Belgian Malinois (14 case, 49 control) and German Shepherd 56 (24 case ve 32 control) were included in the study. 5 genes (CX3CR1, CHST3, OSTF1, IBSP, FGF12) that may be associated with the development of CHD have been determined for molecular genetic analysis. The exon 2 and 3' UTR regions of the CX3CR1 gene were investigated by DNA sequence analysis method and variations were determined. On other genes, it was investigated 5 SNP (CHST3 gene 5? UTR-rs24076746 ve 3? UTR-rs24105396, OSFT1 gene intron 4-rs8995004, IBSP gene intron 3-rs23759713 ve FGF12 gene intron 3-rs851690466) by RFLP method, which were previously reported by different researchers. Determination of haplotypes, determination of miRNA binding sites and the effects of SNP-SNP interactions on the development of CHD disease were investigated by bioinformatics methods. Chi square values, likelihood ratio (OR) and 95% confidence interval (GA) were calculated by logistic regression analysis. No variation was found in the exon 2 region of the CX3CR1 gene, while 8 variations (6 SNP, 1 insersion, 1 deletion) were detected in the 3' UTR region. As a result of the statistical evaluations, it was found that rs9022770 G> A and rs9022769 G> A SNPs were associated with the development of CHD in Belgian Malinois breed dogs (p <0.05). In the other two breeds of dogs, there was no statistically significant relationship between individual variations and the disease. In the Belgian Malinois breed, the G allele for rs9022770 G> A SNP was determined to be the pathogen allele (OR=13,091, %95 GA=2,474-69,264) at the significance level of p = 0.002. For the G> A SNP of rs9022769, the G allele was calculated to be the pathogen allele (OR=3,333, %95 GA=1,279-8,688) at the significance level of p = 0.011. The GT haplotype formed by the CX3CR1 rs9022769 / rs852431030 SNPs was determined to be a pathogen haplotype (OR=6,222, %95 GA=1,280-30,243-0,891) with a significance level of p = 0.022. In other dog breeds investigated, a statistically significant relationship could not be detected between the resulting haplotypes and the development of CHD. Only one of the 5 SNPs that previous researchers reported was associated with the development of CHD, the rs23759713 C> T SNP in the IBSP gene intron 3 was supported by being associated with the development of CHD in German Shepherd Dogs. It was determined that the rs23759713-T allele was a protective allele (OR=0,207, %95 GA=0,048-0,891) at the level of p = 0.026. The other 4 SNPs (rs24076746, rs24105396, rs8995004, rs851690466) have not been associated with the development of CHD. As a result of our research, we think that the 3' UTR polymorphisms (rs9022770, rs9022769) of the CX3CR1 gene, which we found to be associated with the development of CHD, will contribute to marker assised selection (MAS) programs to be applied against CHD disease. The relation of rs23759713 SNP in the intron 3 of the IBSP gene with the development of CHD was also supported in our study. Our research results provide data that can be useful both for illuminating the genetic background of CHD disease and for MAS studies.
Hip dysplasia, a developmental orthopedic disease, occurs with the influence of hereditary and environmental factors. Although it is common in both humans and dogs, the genetic background of the disease has not been elucidated. Although the selection programs based on phenotype against canine hip dysplasia (CHD) have been applied for many years, the desired success has not been achieved. Today, to develop a DNA test that can diagnose hip dysplasia early, the disease causing genetic loci are being identified. In this study, a case-control study was designed to detect genetic variations that may be associated with the development of CHD disease. Breding as service dogs in Turkey, 53 Labrador Retrievers (17 case, 36 control), 63 Belgian Malinois (14 case, 49 control) and German Shepherd 56 (24 case ve 32 control) were included in the study. 5 genes (CX3CR1, CHST3, OSTF1, IBSP, FGF12) that may be associated with the development of CHD have been determined for molecular genetic analysis. The exon 2 and 3' UTR regions of the CX3CR1 gene were investigated by DNA sequence analysis method and variations were determined. On other genes, it was investigated 5 SNP (CHST3 gene 5? UTR-rs24076746 ve 3? UTR-rs24105396, OSFT1 gene intron 4-rs8995004, IBSP gene intron 3-rs23759713 ve FGF12 gene intron 3-rs851690466) by RFLP method, which were previously reported by different researchers. Determination of haplotypes, determination of miRNA binding sites and the effects of SNP-SNP interactions on the development of CHD disease were investigated by bioinformatics methods. Chi square values, likelihood ratio (OR) and 95% confidence interval (GA) were calculated by logistic regression analysis. No variation was found in the exon 2 region of the CX3CR1 gene, while 8 variations (6 SNP, 1 insersion, 1 deletion) were detected in the 3' UTR region. As a result of the statistical evaluations, it was found that rs9022770 G> A and rs9022769 G> A SNPs were associated with the development of CHD in Belgian Malinois breed dogs (p <0.05). In the other two breeds of dogs, there was no statistically significant relationship between individual variations and the disease. In the Belgian Malinois breed, the G allele for rs9022770 G> A SNP was determined to be the pathogen allele (OR=13,091, %95 GA=2,474-69,264) at the significance level of p = 0.002. For the G> A SNP of rs9022769, the G allele was calculated to be the pathogen allele (OR=3,333, %95 GA=1,279-8,688) at the significance level of p = 0.011. The GT haplotype formed by the CX3CR1 rs9022769 / rs852431030 SNPs was determined to be a pathogen haplotype (OR=6,222, %95 GA=1,280-30,243-0,891) with a significance level of p = 0.022. In other dog breeds investigated, a statistically significant relationship could not be detected between the resulting haplotypes and the development of CHD. Only one of the 5 SNPs that previous researchers reported was associated with the development of CHD, the rs23759713 C> T SNP in the IBSP gene intron 3 was supported by being associated with the development of CHD in German Shepherd Dogs. It was determined that the rs23759713-T allele was a protective allele (OR=0,207, %95 GA=0,048-0,891) at the level of p = 0.026. The other 4 SNPs (rs24076746, rs24105396, rs8995004, rs851690466) have not been associated with the development of CHD. As a result of our research, we think that the 3' UTR polymorphisms (rs9022770, rs9022769) of the CX3CR1 gene, which we found to be associated with the development of CHD, will contribute to marker assised selection (MAS) programs to be applied against CHD disease. The relation of rs23759713 SNP in the intron 3 of the IBSP gene with the development of CHD was also supported in our study. Our research results provide data that can be useful both for illuminating the genetic background of CHD disease and for MAS studies.
Açıklama
Anahtar Kelimeler
Köpek kalça displazisi, CX3CR1, IBSP, OSTF1, CHST3, FGF12, FGF12, Polimorfizm, Genetik markır, Canine hip dysplasia, CX3CR1, IBSP, OSTF1, CHST3, FGF12, Polymorphism, Genetic marker
