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Disease detection of collie eye anomaly in shepherd dog breeds
ŽÁKOVÁ, Hana
This thesis deals with the detection of Collie Eye Anomaly (CEA) in Shepherd breeds. CEA is a congenital hereditary eye disease whose main manifestations are choroidal hypoplasia and coloboma of the optic nerve. This disease is incurable and complications such as retinal detachment or intraocular bleeding are often associated with it. The development of this anomaly is caused by a deletion of the 7.8 kb region in intron 4 of the NHEJ1 gene. The aim of this thesis is to optimize PCR and real-time PCR methods for the detection of CEA causal mutation and subsequent screening of DNA samples from Shepherd breeds dogs. The investigated group consisted of 138 DNA samples collected from individuals of the Border Collie, Smooth Collie, Rough Collie, Sheltie, Australian Shepherd and German Shepherd breeds. Biological materials were obtained via buccal swab. Both examined methods yielded the same results with regards to the subjects' genotypes. The screening of samples confirmed the findings of previous research, that the highest incidence of CEA occurs in Rough Collie (91%) and Sheltie (23%) breeds, respectively. The lowest incidence of recessive homozygotes (4%) was found in the Border Collie breed. Among other breeds, no dogs affected by CEA were detected.
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Detection of mutation HSF4 causing hereditary cataract in sheep dogs
VOKÁLKOVÁ, Lenka
Hereditary cataract belongs amongs the most common diseases of the eye with the shepherd dog breeds. It is a very serious disease, which causes the deterioration of sight of the dog. With the neglection can the illness cause even blindness. Heteritary cataract manifests itself with bilateral turbidity of the eye. Course of the illness differs and the beginning of the sickness can start at any age of the dog. The formation of the cataract can be the result of another eye disease or the in jury of the eye. In this case it is a secondary cataract.Primary cataract is directed by genes. Candidate gene for the formation of the hereditary cataract is the HSF4 gene. This gene include an important role in the immunity system. It encodes proteins of the thermal shock, that protects the cell from adverse effects of the outer environment, like high temperatures or UV rays. Besides that it also cantributes to the development of the eye lens fibers. Mutation in the exon of the gene HSF4 causes the degradation of these fibers, hence cataract. Hereditary cataract is a serious illness, which must be cured by surgical implantation of the eye lens. Therefore, timely diagnosis and decommission of affected individuals from the breed, that the transfer of the ilness to the next generation will be prevented. I deal with the detection of a mutation in the HSF4 gene causing cataracts in my diploma thesis. I have chosen the fragment analysis as the most suitable method for detection of the mutation in the gene HSF4. The mutation is in the insertion of one nucleotid C. Fragment analysis can determine very precisely the mass of the DNA molecule and permits to differ even a fragment, which differs only in one base. In the practical part of the thesis were examined 100 samples of the dogs of breeds australian shepperd, border collie, german shepherd and long ans short haired collie. Only one of the dogs was evaluated as the carrier of the hereditary cataract. It was the dog of the australian shepperd breed. Three of the samples were non evaluable, caused by wrong sampling or degradation of the DNA. The rest of the evaluated dogs (96 samples) were healty.
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Detection MDR1 mutation in shepherd dog breeds by loop mediated isothermal amplification and restriction analysis
VYHLÍDALOVÁ, Nikola
Multiple drug resistance is caused by MDR1 gene mutation. It is a common genetic disease of Shepherd Dogs from which Collie breed is the most affected. Described mutation is caused by deletion of four nucleotides, which leads to a frameshift and premature termination of synthesis of the P-glycoprotein, one of the important transport proteins of some drugs. Synthesized non-functional P-glycoprotein is unable to eliminate drugs from the brain, which leads to a neurotoxic problems in affected dogs. The theoretical part describes structure and function of P-glycoprotein and its inhibition caused by MDR1 coding gene mutation. The most commonly affected breeds are subjected to analysis as well as the problem of drugs for dogs with this mutation. Review also includes a description and principle of methods used for detecting mutation of MDR1 gene. The aim of this thesis was to optimize and screen DNA samples of Shepherd Dogs for the MDR1 gene mutation by LAMP and PCR-RFLP as well as by mutual comparison of both methods. The empirical part presents the results of the study. The studied population consisted about 140 samples of Shepherd Dogs DNA. Our results confirmed the highest incidence of recessive homozygotes in the tested population of Smooth Collie (25 %). Moreover, the number of heterozygous was also the highest in this breed (50 %). However, the number of heterozygous dogs in the tested population of German Shepherd was surprisingly comparable with population of Smooth Collie, because it reached 49 %. Equal number of the German Shepherd was dominantly homozygous. Whereas, the highest number of healthy dogs was found in the tested population of Border Collie breed (65 %).
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