![ABSTRACT Epilepsy is a brain disorder of abnormal electrical signals that cause seizures. It varies in severity and is common in children and adults. Idiopathic Epilepsy involves recurrent seizures in which no underlying abnormality can be identified (Patterson et al. 2003). Each year 200,000 new human cases are diagnosed, being the second most common neurological condition. Some studies suggest that ion channel genes cause epilepsy for many of the human forms. The known human mutations have not shown to be the cause in four dog breeds (Ekenstedt et al. 2011). Breeds such as Bernese Mountain dogs, Vizlas, Labrador Retrievers and Australian Shepherds have a high incidence of epilepsy. For this study, Australian Shepherds were chosen because of their high frequency of epilepsy that appears to be inherited based on pedigree analysis. As generations pass, there are some genes in close physical proximity on the same chromosome that do not recombine and are in what is called linkage disequilibrium (LD). In regions of LD there is a haplotype that is shared between closely related breeds and common ancestors. Long LD allows many fewer genetic markers and cases to be used for genome wide association studies in dogs compared to people (Sutter et al. 2004). Breeds that have passed through population bottlenecks are prone to carry more recessive traits since inbreeding is frequently employed. As a result, inbreeding may be responsible for common diseases in these breeds. Diseases include cancer, epilepsy, deafness and heart diseases. Whole genome association (WGA) is a technique that examines the genome of different individuals searching for differences among them. DNA samples are placed on a crystal chip that genotypes 170,000 canine SNPs. A SNP is a single nucleotide polymorphism, a difference in base pairs when a single nucleotide is altered at a specific location. The lab team performed previous work (WGA) for 40 Australian Shepherds in which they established that there is a possibility of one or two genes that cause epilepsy in this breed. These genes are likely to be at specific locations on canine chromosomes 1 (CFA1) and/or CFA19, based on Chi-square analysis of the data and the resulting p-values. To obtain this data, they tested 19 affected dogs and 21 unaffected. CFA1 and CFA19 showed a number of SNPs close together that stand out (Figure 1) when graphed on a negative logarithmic scale of their Chi-square p-value (-log p-value). In chromosome 19, one of the associated SNPs is between nucleotides G and A, and G is strongly associated with epilepsy. Chromosome 1 has one of the associated SNPs between C and T, with C being strongly associated with epilepsy. The SNP BICF2G63036912 in CFA19 is located at 13,956,701 bp of the chromosome and the SNP BICF2G630711334 in CFA1 is located at 12,632,317 bps. On the canine SNP array, there is on average one SNP every 14,000bp in the total of 2.4 billion bps of the canine genome. DOK6 is a gene located in chromosome 1 that is very near some of the associated SNP markers. It has 8 coding exons. Our objective was to perform Chi-square analysis for 88 additional Australian Shepherds for two of the associated SNPs to determine if the original association was real or only by chance. A second objective was to determine if there are any coding mutations in DOK6 for epilepsy in Australian Shepherds. The overall hypothesis of this work is that there is one or more epilepsy causing mutations in these specific regions of canine chromosomes 1 and/or 19. INTRODUCTION MATERIALS AND METHODS RESULTS Dog DNA samples were collected along with their medical information. Affected dogs must have had their first seizure before 6 years, whereas normal dogs were at least 8 years without a history of seizures. In order for them to be included in this study, they must have fulfilled these requirements: 1. Two or more seizures 2. Normal in between of seizures 3. Normal blood panels (no abnormalities). Based on the WGA results, one dog of each genotype was selected to serve as a control for the enzyme digest. For CFA 19, the genotypes were homozygous AA, heterozygous AG, and homozygous GG. For CFA1, the genotypes were homozygous TT, heterozygous CT, and homozygous CC. These 6 control samples served as references when determining the genotypes of an additional 88 dogs via RFLP. SNPs were chosen based on p-value and availability of RFLP. Fifteen ng of purified DNA, specific 20 µM forward and reverse primers (1.5uL), 300 µM dNTPs (1.5uL), 10x buffer (1.5uL) in water (5.6uL), and TAQ polymerase (0. 5 uL) were used for PCR to amplify the region containing each SNP. Cycling conditions were: 95 ° C for 15 min, 35 cycles of 95 ° C for 30 s, 60 ° C for 30 s, and 72 ° C for 30 s; and 72 ° C for 15 min. The PCR bands of 385 bp for both CFA1 and CFA19 were visualized on a 2% agarose gel. Enzymes Sau3AI (8u) and BsmI (5u) were used for each of the 2 chromosomes of interests. Sau3AI (CFA1) recognizes nucleotide C and cuts there when the sequence T G A T C is present. Where as BsmI (CFA19) recognizes nucleotide G and will cut there every time the sequence G C A T C is present. For the CFA1 or CFA19 SNP respectively, the resulting banding patterns were visualized on a 2% agarose gel as follows: 385bp for homozygous TT or AA; 385, 216 and 169 bp for heterozygous CT or AG, and 216bp and 169bp for homozygous CC or GG. The RFLP results determined the genotype of the additional 88 dogs’ samples leading the way for the statistical Chi-square analysis to verify the association found by the prior WGA study. Additionally, DOK6 sequencing was completed in one affected and one unaffected dog. As described above, PCR was done with primers that encompassed all 8 exons. Sanger sequencing was then performed, and the results were compared between the two dogs and against the published dog sequence. Figure 2. Gel electrophoresis results. Shows the 3 different band sizes after performing the RFLP test for CFA19. The last three samples are the controls for homozygous AA, heterozygous AG, and homozygous GG. For this chromosome, G is strongly associated with epilepsy; therefore, it is hypothesized that affected dogs most often have a genotype of GG in the studied position of 13,956,701bp. CONCLUSIONS REFERENCES Acknowledgements I want to give special thanks to my mentor Dr. Ned Patterson and Katie Minor for all their effort in guiding me during my summer research project. Also, I want to thank Dr. Eva Furrow for her help with Chi-square analysis. Funded by: AKC Canine Health Foundation LSSURP RISE program R25GM059429-12 Idiopathic epilepsy implies having recurrent seizures in which no anomaly is known. It is a common disorder that affects about 4% of dogs and 1% of people. Previous Whole Genome Analysis showed statistical association of single nucleotide polymorphism (SNP) associations with Epilepsy in a study of 40 Australian shepherd dogs (Aussies) on loci of CFA1 and CFA19. This research further evaluated SNPs between epileptic and non-epileptic Aussies. The major objectives of this portion of the study were to confirm the statistical associations observed on CFA1 and CFA19 in additional case and control Aussies via PCR-RFLP tests and to sequence one candidate gene, DOK6. Two PCR-RFLP SNP tests were performed on 88 additional Aussies to compare their genotype frequencies by Chi-square statistical analysis. Sequencing of DOK6 was performed on one case and one control Aussie. By studying the SNPs in Aussies, we confirmed different genotype frequencies between normal and affected dogs on canine chromosomes 1 and 19 indicating possible causative genes in each location. Sequencing of DOK6 did not show any polymorphisms in the exons (coding regions). Ekenstedt K, Patterson E, Minor K et al. 2011. Candidate genes for idiopathic epilepsy in four dog breeds BMC Genetics [Internet]. Available from: http://www.biomedcentral.com/1471-2156/12/38 Patterson E, Mickelson J, Da Y et al. 2003. Clinical Characteristics and Inheritance of Idiopathic Epilepsy in Vizlas. Journal of Internal Veterinary Medicine Volume 17, Issue 3, pages 319-325, May 2003. [Internet]. Available from: http://onlinelibrary.wiley.com/doi/10.1111/j.1939-1676.2003.tb02455.x/abstract Sutter N, Eberle M, Parker H et al. 2004. Extensive and breed-specific linkage disequilibrium in Canis familiaris. [Internet]. Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC534662 / AA AG GG Significant associations of SNPs between affected and unaffected Aussies were still observed on CFA1 and CFA19 with the addition of (23) cases and (65) controls, for a total of (42) cases and (86) controls. The G allele in CFA19 (SNP - BICF2G63036912) is strongly associated with epilepsy and has a frequency of 47.3% in affected dogs. In CFA1 (SNP - BICF2G630711334), the frequency for the C allele is also associated with epilepsy and has a frequency of 51.3% in affected dogs. The p-values for association for these two SNP with epilepsy were 0.0103 and 0.000750 for CFA1 and CFA19, respectively. These results strongly support our hypothesis of a causative mutation near these locations in CFA1 and CFA19. Affected dogs are more likely to have G allele for the CFA19 SNP and the C allele for the CFA1 SNP as shown in Figure 3. Sanger sequence of 8 exons of DOK6 did not reveal any differences in the case versus the control Aussie selected. Epilepsy in Australian Shepherds is suspected to be an inherited disease and prior work has shown an association between loci on CFA1 and CFA19 and this seizure disorder. By testing 23 affected and 65 unaffected Australian Shepherd dogs for 2 different RFLP SNPs and comparing genotypes, the association of the loci on both CFA1 and CFA19 with epilepsy was confirmed. It is concluded that there are likely genetic mutations in both regions that contribute to the development of epilepsy in Australian Shepherds. Future work could involve WGA in closely related breed such as Border Collies. Based on previous research on Vizlas and English Springer Spaniels, the lab team may continue their investigation focusing on these 2 chromosomal areas. A SNP Chi-square analysis could serve to compare between breeds and determine if these associations are shared across breeds. In addition, new research may involve looking at the other genes within 1-2 million base pairs near DOK6 to evaluate for a causative mutation for epilepsy in the breed. Figure 1. Results of a WGA study with 19 dogs affected by epilepsy and 21 unaffected. Shows SNPs that stand out when graphed on a negative logarithmic scale of their Chi-square p-value (-log p-value) on the y-axis and the canine chromosomes in order on the x-axis . These are in CFA1 ( pink ) and in CFA19 (green). CFA19 has one that is statistically significant being over 1.3, equivalent to a p-value less than 0.05. 385bp 216bp 169bp CFA19 AA AG GG Figure 3. CFA1 showed statistical significance with a greater C allele frequency for affected dogs. CFA19 showed statistical significance with a greater G allele frequency for affected dogs. Epilepsy in Australian Shepherd Dogs Valeria Rivera 1,2 , Katie M. Minor B.A 3 ; Eva Furrow V.M.D., Edward (Ned) Patterson D.V.M., Ph.D. 3 University of Minnesota, Life Sciences Summer Undergraduate Research Program : Heart, Lung & Blood University of Puerto Rico at Cayey 3. Department of Veterinary Clinical Sciences, University of Minnesota College of Veterinary Medicine, St. Paul, MN.](https://image.slidesharecdn.com/riveravaleriasame-110924210801-phpapp01/75/Rivera-valeria-same-1-2048.jpg)
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- 1. ABSTRACT Epilepsy isa brain disorder of abnormal electrical signals that cause seizures. It varies in severity and is common in children and adults. Idiopathic Epilepsy involves recurrent seizures in which no underlying abnormality can be identified (Patterson et al. 2003). Each year 200,000 new human cases are diagnosed, being the second most common neurological condition. Some studies suggest that ion channel genes cause epilepsy for many of the human forms. The known human mutations have not shown to be the cause in four dog breeds (Ekenstedt et al. 2011). Breeds such as Bernese Mountain dogs, Vizlas, Labrador Retrievers and Australian Shepherds have a high incidence of epilepsy. For this study, Australian Shepherds were chosen because of their high frequency of epilepsy that appears to be inherited based on pedigree analysis. As generations pass, there are some genes in close physical proximity on the same chromosome that do not recombine and are in what is called linkage disequilibrium (LD). In regions of LD there is a haplotype that is shared between closely related breeds and common ancestors. Long LD allows many fewer genetic markers and cases to be used for genome wide association studies in dogs compared to people (Sutter et al. 2004). Breeds that have passed through population bottlenecks are prone to carry more recessive traits since inbreeding is frequently employed. As a result, inbreeding may be responsible for common diseases in these breeds. Diseases include cancer, epilepsy, deafness and heart diseases. Whole genome association (WGA) is a technique that examines the genome of different individuals searching for differences among them. DNA samples are placed on a crystal chip that genotypes 170,000 canine SNPs. A SNP is a single nucleotide polymorphism, a difference in base pairs when a single nucleotide is altered at a specific location. The lab team performed previous work (WGA) for 40 Australian Shepherds in which they established that there is a possibility of one or two genes that cause epilepsy in this breed. These genes are likely to be at specific locations on canine chromosomes 1 (CFA1) and/or CFA19, based on Chi-square analysis of the data and the resulting p-values. To obtain this data, they tested 19 affected dogs and 21 unaffected. CFA1 and CFA19 showed a number of SNPs close together that stand out (Figure 1) when graphed on a negative logarithmic scale of their Chi-square p-value (-log p-value). In chromosome 19, one of the associated SNPs is between nucleotides G and A, and G is strongly associated with epilepsy. Chromosome 1 has one of the associated SNPs between C and T, with C being strongly associated with epilepsy. The SNP BICF2G63036912 in CFA19 is located at 13,956,701 bp of the chromosome and the SNP BICF2G630711334 in CFA1 is located at 12,632,317 bps. On the canine SNP array, there is on average one SNP every 14,000bp in the total of 2.4 billion bps of the canine genome. DOK6 is a gene located in chromosome 1 that is very near some of the associated SNP markers. It has 8 coding exons. Our objective was to perform Chi-square analysis for 88 additional Australian Shepherds for two of the associated SNPs to determine if the original association was real or only by chance. A second objective was to determine if there are any coding mutations in DOK6 for epilepsy in Australian Shepherds. The overall hypothesis of this work is that there is one or more epilepsy causing mutations in these specific regions of canine chromosomes 1 and/or 19. INTRODUCTION MATERIALS AND METHODS RESULTS Dog DNA samples were collected along with their medical information. Affected dogs must have had their first seizure before 6 years, whereas normal dogs were at least 8 years without a history of seizures. In order for them to be included in this study, they must have fulfilled these requirements: 1. Two or more seizures 2. Normal in between of seizures 3. Normal blood panels (no abnormalities). Based on the WGA results, one dog of each genotype was selected to serve as a control for the enzyme digest. For CFA 19, the genotypes were homozygous AA, heterozygous AG, and homozygous GG. For CFA1, the genotypes were homozygous TT, heterozygous CT, and homozygous CC. These 6 control samples served as references when determining the genotypes of an additional 88 dogs via RFLP. SNPs were chosen based on p-value and availability of RFLP. Fifteen ng of purified DNA, specific 20 µM forward and reverse primers (1.5uL), 300 µM dNTPs (1.5uL), 10x buffer (1.5uL) in water (5.6uL), and TAQ polymerase (0. 5 uL) were used for PCR to amplify the region containing each SNP. Cycling conditions were: 95 ° C for 15 min, 35 cycles of 95 ° C for 30 s, 60 ° C for 30 s, and 72 ° C for 30 s; and 72 ° C for 15 min. The PCR bands of 385 bp for both CFA1 and CFA19 were visualized on a 2% agarose gel. Enzymes Sau3AI (8u) and BsmI (5u) were used for each of the 2 chromosomes of interests. Sau3AI (CFA1) recognizes nucleotide C and cuts there when the sequence T G A T C is present. Where as BsmI (CFA19) recognizes nucleotide G and will cut there every time the sequence G C A T C is present. For the CFA1 or CFA19 SNP respectively, the resulting banding patterns were visualized on a 2% agarose gel as follows: 385bp for homozygous TT or AA; 385, 216 and 169 bp for heterozygous CT or AG, and 216bp and 169bp for homozygous CC or GG. The RFLP results determined the genotype of the additional 88 dogs’ samples leading the way for the statistical Chi-square analysis to verify the association found by the prior WGA study. Additionally, DOK6 sequencing was completed in one affected and one unaffected dog. As described above, PCR was done with primers that encompassed all 8 exons. Sanger sequencing was then performed, and the results were compared between the two dogs and against the published dog sequence. Figure 2. Gel electrophoresis results. Shows the 3 different band sizes after performing the RFLP test for CFA19. The last three samples are the controls for homozygous AA, heterozygous AG, and homozygous GG. For this chromosome, G is strongly associated with epilepsy; therefore, it is hypothesized that affected dogs most often have a genotype of GG in the studied position of 13,956,701bp. CONCLUSIONS REFERENCES Acknowledgements I want to give special thanks to my mentor Dr. Ned Patterson and Katie Minor for all their effort in guiding me during my summer research project. Also, I want to thank Dr. Eva Furrow for her help with Chi-square analysis. Funded by: AKC Canine Health Foundation LSSURP RISE program R25GM059429-12 Idiopathic epilepsy implies having recurrent seizures in which no anomaly is known. It is a common disorder that affects about 4% of dogs and 1% of people. Previous Whole Genome Analysis showed statistical association of single nucleotide polymorphism (SNP) associations with Epilepsy in a study of 40 Australian shepherd dogs (Aussies) on loci of CFA1 and CFA19. This research further evaluated SNPs between epileptic and non-epileptic Aussies. The major objectives of this portion of the study were to confirm the statistical associations observed on CFA1 and CFA19 in additional case and control Aussies via PCR-RFLP tests and to sequence one candidate gene, DOK6. Two PCR-RFLP SNP tests were performed on 88 additional Aussies to compare their genotype frequencies by Chi-square statistical analysis. Sequencing of DOK6 was performed on one case and one control Aussie. By studying the SNPs in Aussies, we confirmed different genotype frequencies between normal and affected dogs on canine chromosomes 1 and 19 indicating possible causative genes in each location. Sequencing of DOK6 did not show any polymorphisms in the exons (coding regions). Ekenstedt K, Patterson E, Minor K et al. 2011. Candidate genes for idiopathic epilepsy in four dog breeds BMC Genetics [Internet]. Available from: http://www.biomedcentral.com/1471-2156/12/38 Patterson E, Mickelson J, Da Y et al. 2003. Clinical Characteristics and Inheritance of Idiopathic Epilepsy in Vizlas. Journal of Internal Veterinary Medicine Volume 17, Issue 3, pages 319-325, May 2003. [Internet]. Available from: http://onlinelibrary.wiley.com/doi/10.1111/j.1939-1676.2003.tb02455.x/abstract Sutter N, Eberle M, Parker H et al. 2004. Extensive and breed-specific linkage disequilibrium in Canis familiaris. [Internet]. Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC534662 / AA AG GG Significant associations of SNPs between affected and unaffected Aussies were still observed on CFA1 and CFA19 with the addition of (23) cases and (65) controls, for a total of (42) cases and (86) controls. The G allele in CFA19 (SNP - BICF2G63036912) is strongly associated with epilepsy and has a frequency of 47.3% in affected dogs. In CFA1 (SNP - BICF2G630711334), the frequency for the C allele is also associated with epilepsy and has a frequency of 51.3% in affected dogs. The p-values for association for these two SNP with epilepsy were 0.0103 and 0.000750 for CFA1 and CFA19, respectively. These results strongly support our hypothesis of a causative mutation near these locations in CFA1 and CFA19. Affected dogs are more likely to have G allele for the CFA19 SNP and the C allele for the CFA1 SNP as shown in Figure 3. Sanger sequence of 8 exons of DOK6 did not reveal any differences in the case versus the control Aussie selected. Epilepsy in Australian Shepherds is suspected to be an inherited disease and prior work has shown an association between loci on CFA1 and CFA19 and this seizure disorder. By testing 23 affected and 65 unaffected Australian Shepherd dogs for 2 different RFLP SNPs and comparing genotypes, the association of the loci on both CFA1 and CFA19 with epilepsy was confirmed. It is concluded that there are likely genetic mutations in both regions that contribute to the development of epilepsy in Australian Shepherds. Future work could involve WGA in closely related breed such as Border Collies. Based on previous research on Vizlas and English Springer Spaniels, the lab team may continue their investigation focusing on these 2 chromosomal areas. A SNP Chi-square analysis could serve to compare between breeds and determine if these associations are shared across breeds. In addition, new research may involve looking at the other genes within 1-2 million base pairs near DOK6 to evaluate for a causative mutation for epilepsy in the breed. Figure 1. Results of a WGA study with 19 dogs affected by epilepsy and 21 unaffected. Shows SNPs that stand out when graphed on a negative logarithmic scale of their Chi-square p-value (-log p-value) on the y-axis and the canine chromosomes in order on the x-axis . These are in CFA1 ( pink ) and in CFA19 (green). CFA19 has one that is statistically significant being over 1.3, equivalent to a p-value less than 0.05. 385bp 216bp 169bp CFA19 AA AG GG Figure 3. CFA1 showed statistical significance with a greater C allele frequency for affected dogs. CFA19 showed statistical significance with a greater G allele frequency for affected dogs. Epilepsy in Australian Shepherd Dogs Valeria Rivera 1,2 , Katie M. Minor B.A 3 ; Eva Furrow V.M.D., Edward (Ned) Patterson D.V.M., Ph.D. 3 University of Minnesota, Life Sciences Summer Undergraduate Research Program : Heart, Lung & Blood University of Puerto Rico at Cayey 3. Department of Veterinary Clinical Sciences, University of Minnesota College of Veterinary Medicine, St. Paul, MN.
Editor's Notes
- #2 Background photo CFA1 showed no statistical significance when looking at the chi-square results. CFA19 showed statistical significance