The epilepsy paper 3


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The epilepsy paper 3

  1. 1. Epilepsy in Australian Shepherd Dogs Valeria Rivera1,2, Katie M. Minor B.A3; Eva Furrow V.M.D., Edward (Ned) Patterson D.V.M., Ph.D.3 1.University of Minnesota, Life Sciences Summer Undergraduate Research Program: Heart, Lung, & Blood 2.University of Puerto Rico at Cayey 3. Department of Veterinary Clinical Sciences, University of Minnesota College of Veterinary Medicine, St. Paul, MN. Abstract 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) 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). Key words: Epilepsy, canines, genetic basis, SNPs, and Australian Shepherds. condition. Some studies suggest that ionIntroduction channel genes cause epilepsy for many of the human forms. The known human Epilepsy is a brain disorder of mutations have not shown to be the causeabnormal electrical signals that cause in four dog breeds (Ekenstedt et al. 2011).seizures. It varies in severity and is Breeds such as Bernese Mountain dogs,common in children and adults. Vizslas, Labrador Retrievers, andIdiopathic Epilepsy (IE) involves Australian Shepherds have a highrecurrent seizures in which no underlying incidence of epilepsy. For this study,abnormality can be identified (Patterson Australian Shepherds were chosenet al. 2003). Each year 200,000 new because of their high frequency ofhuman cases are diagnosed, being the epilepsy that appears to be inheritedsecond most common neurological based on pedigree analysis. As 1
  2. 2. generations pass, there are some genes in G is strongly associated with epilepsy.close physical proximity on the same Chromosome 1 has one of the associatedchromosome that do not recombine and SNPs between C and T, with C beingare in what is called linkage strongly associated with epilepsy. Thedisequilibrium (LD). In regions of LD SNP BICF2G63036912 on CFA19 isthere is a haplotype that is shared located at 13,956,701 bp of thebetween closely related breeds and chromosome and the SNPcommon ancestors. Long LD allows many BICF2G630711334 on CFA1 is located atfewer genetic markers and cases to be 12,632,317 bps. On the canine SNP array,used for genome wide association studies there is on average one SNP everyin dogs compared to people (Sutter et al. 14,000bp in the total of 2.4 billion bps of2004). Breeds that have passed through the canine genome.population bottlenecks are prone to carry DOK6 is a gene located inmore recessive traits since inbreeding is chromosome 1 that is very near some offrequently employed. As a result, the associated SNP markers. It has 8inbreeding may be responsible for coding exons. Our objective was tocommon diseases in these breeds. perform Chi-square analysis for 88Diseases include cancer, epilepsy, additional Australian Shepherds for twodeafness, and heart diseases. of the associated SNPs to determine if the Whole genome association (WGA) original association was real or only byis a technique that examines the genome chance. A second objective was toof different individuals searching for determine if there are any codingdifferences among them. DNA samples mutations in DOK6 for epilepsy inare placed on a crystal chip that Australian Shepherds. The overallgenotypes 170,000 canine SNPs. A SNP is hypothesis of this work is that there isa single nucleotide polymorphism, a one or more epilepsy causing mutationsdifference in base pairs when a single in these specific regions of caninenucleotide is altered at a specific location. chromosomes 1 and/or 19.The lab team performed previous work(WGA) for 40 Australian Shepherds in Figure 1: Aussies Chi-square testwhich they established that there is apossibility of one or two genes that causeepilepsy in this breed. These genes are likely to be atspecific locations on canine chromosomes1 (CFA1) and/or CFA19, based on Chi-square analysis of the data and theresulting p-values. To obtain this data,they tested 19 affected dogs and 21unaffected. CFA1 and CFA19 showed anumber of SNPs close together that standout (Figure 1) when graphed on a Figure 1. Results of a WGA study with 19 dogsnegative logarithmic scale of their Chi- affected by epilepsy and 21 unaffected. Shows SNPssquare p-value (-log p-value). In that stand out when graphed on a negativechromosome 19, one of the associated logarithmic scale of their Chi-square p-value (-log p-SNPs is between nucleotides G and A, and value) on the y-axis and the canine chromosomes in 2
  3. 3. order on the x-axis. These are in CFA1 (pink) and in when the sequence T G A T C is present.CFA19 (green). CFA19 has one that is statistically Where as BsmI (CFA19) recognizessignificant being over 1.3, equivalent to a p-valueless than 0.05. nucleotide G and will cut there every time the sequence G C A T C is present. ForMaterials and Methods CFA1 RFLP, 8 units (2µL) of sau3AI were used along with NEB 1(2µL), BSA (0.2µL), Dog DNA samples were collected and water (10.8µL). The 8 unit masteralong with their medical information. mix (15µL) was pipetted into PCRAffected dogs must have had their first product to digest overnight at 37°C hold.seizure before 6 years, whereas normal For CFA19, 5 units of the enzyme BmsIdogs were at least 8 years without a (0.5µL) were used leaving it digest for 3history of seizures. In order for them to hours. The resulting banding patternsbe included in this study, they must have were visualized on a 2% agarose gel asfulfilled these requirements: 1. Two or follows: 385bp for homozygous TT or AA;more seizures 2. Normal in between of 385, 216 and 169 bp for heterozygous CTseizures 3. Normal blood panels (no or AG, and 216bp and 169bp forabnormalities). homozygous CC or GG for CFA1 and Based on the WGA results, one dog CFA19 respectively. The RFLP resultsof each genotype was selected to serve as determined the genotype of the additionala control for the enzyme digest. For CFA 88 dogs’ samples leading the way for the19, the genotypes were homozygous AA, statistical Chi-square analysis to verifyheterozygous AG, and homozygous GG. the association found by the prior WGAFor CFA1, the genotypes were study.homozygous TT, heterozygous CT, and Additionally, DOK6 sequencinghomozygous CC. These 6 control samples was completed in one affected and oneserved as references when determining unaffected dog. As described above, PCRthe genotypes of an additional 88 dogs via was done with primers that encompassedRFLP. SNPs were chosen based on p-value all 8 exons. Sanger sequencing was thenand availability of RFLP. performed, and the results were Fifteen ng of purified DNA, specific compared between the two dogs and20 µM forward and reverse primers against the published dog sequence.(1.5uL), 300 µM dNTPs (1.5µL), 10xbuffer (1.5µL) in water (5.6µL), and TAQ Figure 2: RFLP for CFA19polymerase (0. 5 µL) were used for PCR toamplify 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 CFA1and CFA19 were visualized on a 2%agarose gel. Enzymes Sau3AI (8u) and BsmI(5u) were used for each of the 2chromosomes of interests. Sau3AI (CFA1)recognizes nucleotide C and cuts there Figure 2 Shows the 3 different band sizes after 3
  4. 4. performing the RFLP test for CFA19. The last three greater C allele frequency for affected dogs.samples are the controls for homozygous AA • CFA19 showed statistical significance withheterozygous AG and homozygous GG. For this a greater G allele frequency for affectedchromosome, G is strongly associated with dogs.epilepsy; therefore, it is hypothesized that affected Conclusiondogs have most often have a genotype of GG in thestudied position of 13,956,701bp. Epilepsy in Australian Shepherds is suspected to be an inherited diseaseResults and prior work has shown an association between loci on CFA1 and CFA19 in this Significant associations of SNPs seizure disorder. By testing 23 affectedbetween affected and unaffected Aussies and 65 unaffected Australian Shepherdwere still observed on CFA1 and CFA19 dogs for 2 different RFLP SNPs andwith the addition of (23) cases and (65) comparing genotypes, the association ofcontrols, for a total of (42) cases and (86) the loci on both CFA1 and CFA19 withcontrols. The G allele in CFA19 (SNP - epilepsy was confirmed. It is concludedBICF2G63036912) is strongly associated that there are likely genetic mutations inwith epilepsy and has a frequency of both regions that contribute to the47.3% in affected dogs. In CFA1 (SNP - development of epilepsy in AustralianBICF2G630711334), the frequency for the Shepherds.C allele is also associated with epilepsy Future work could involve WGA inand has a frequency of 51.3% in affected closely related breed such as Borderdogs. Collies. Based on previous research on The p-values for association for Vizslas and English Springer Spaniels, thethese two SNP with epilepsy were 0.0103 lab team may continue their investigationand 0.000750 for CFA1 and CFA19, focusing on these 2 chromosomal areas. Arespectively. These results strongly SNP Chi-square analysis could serve tosupport our hypothesis of a causative compare between breeds and determinemutation near these locations in CFA1 if these associations are shared acrossand CFA19. Affected dogs are more likely breeds. In addition, new research mayto have G allele for the CFA19 SNP and the involve looking at the other genes withinC allele for the CFA1 SNP as shown in 1-2 million base pairs near DOK6 toFigure 3. evaluate for a causative mutation for Sanger sequence of 8 exons of DOK6 epilepsy in the breed.did not reveal any differences in the caseversus the control Aussie selected. Acknowledgements I want to give special thanks to myFigure: 3 Allele frequencies 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 FoundationFigure 3. • CFA1 showed statistical significance with a LSSURP 4
  5. 5. RISE program R25GM059429-12References1. Ekenstedt k, Patterson E, Minor K et al.2011. Candidate genes for idiopathicepilepsy in four dog breeds BMC Genetics[Internet]. Available from: Patterson E, Mickelson J, Da Y et al.2003. Clinical Characteristics andInheritance of Idiopathic Epilepsy inVizlas. Journal of Internal VeterinaryMedicine Volume 17, Issue 3, pages319-325, May 2003. [Internet]. Availablefrom: Sutter N, Eberle M, Parker H et al.2004.Extensive and breed-specificlinkage disequilibrium in Canis familiaris.[Internet]. Available from: 5