The study confirms associations between epilepsy in Australian Shepherd dogs and two locations on chromosomes 1 (CFA1) and 19 (CFA19) found in previous research. PCR-RFLP tests on 88 additional dogs showed different genotype frequencies between normal and affected dogs, supporting genes in these locations that may cause epilepsy. Sequencing of the DOK6 gene near associated SNPs on CFA1 found no differences between one affected and unaffected dog. The results support the hypothesis that mutations in these chromosomal regions contribute to epilepsy in Australian Shepherds.

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 ion
Introduction 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 cause
abnormal 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, and
Idiopathic Epilepsy (IE) involves Australian Shepherds have a high
recurrent seizures in which no underlying incidence of epilepsy. For this study,
abnormality can be identified (Patterson Australian Shepherds were chosen
et al. 2003). Each year 200,000 new because of their high frequency of
human cases are diagnosed, being the epilepsy that appears to be inherited
second most common neurological based on pedigree analysis. As
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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 associated
chromosome that do not recombine and SNPs between C and T, with C being
are in what is called linkage strongly associated with epilepsy. The
disequilibrium (LD). In regions of LD SNP BICF2G63036912 on CFA19 is
there is a haplotype that is shared located at 13,956,701 bp of the
between closely related breeds and chromosome and the SNP
common ancestors. Long LD allows many BICF2G630711334 on CFA1 is located at
fewer 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 every
in dogs compared to people (Sutter et al. 14,000bp in the total of 2.4 billion bps of
2004). Breeds that have passed through the canine genome.
population bottlenecks are prone to carry DOK6 is a gene located in
more recessive traits since inbreeding is chromosome 1 that is very near some of
frequently employed. As a result, the associated SNP markers. It has 8
inbreeding may be responsible for coding exons. Our objective was to
common diseases in these breeds. perform Chi-square analysis for 88
Diseases include cancer, epilepsy, additional Australian Shepherds for two
deafness, and heart diseases. of the associated SNPs to determine if the
Whole genome association (WGA) original association was real or only by
is a technique that examines the genome chance. A second objective was to
of different individuals searching for determine if there are any coding
differences among them. DNA samples mutations in DOK6 for epilepsy in
are placed on a crystal chip that Australian Shepherds. The overall
genotypes 170,000 canine SNPs. A SNP is hypothesis of this work is that there is
a single nucleotide polymorphism, a one or more epilepsy causing mutations
difference in base pairs when a single in these specific regions of canine
nucleotide 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 test
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 Figure 1. Results of a WGA study with 19 dogs
negative logarithmic scale of their Chi- affected by epilepsy and 21 unaffected. Shows SNPs
square p-value (-log p-value). In that stand out when graphed on a negative
chromosome 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. 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) recognizes
significant being over 1.3, equivalent to a p-value
less than 0.05. nucleotide G and will cut there every time
the sequence G C A T C is present. For
Materials 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 master
along with their medical information. mix (15µL) was pipetted into PCR
Affected 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 BmsI
dogs were at least 8 years without a (0.5µL) were used leaving it digest for 3
history of seizures. In order for them to hours. The resulting banding patterns
be included in this study, they must have were visualized on a 2% agarose gel as
fulfilled 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 CT
seizures 3. Normal blood panels (no or AG, and 216bp and 169bp for
abnormalities). homozygous CC or GG for CFA1 and
Based on the WGA results, one dog CFA19 respectively. The RFLP results
of each genotype was selected to serve as determined the genotype of the additional
a control for the enzyme digest. For CFA 88 dogs’ samples leading the way for the
19, the genotypes were homozygous AA, statistical Chi-square analysis to verify
heterozygous AG, and homozygous GG. the association found by the prior WGA
For CFA1, the genotypes were study.
homozygous TT, heterozygous CT, and Additionally, DOK6 sequencing
homozygous CC. These 6 control samples was completed in one affected and one
served as references when determining unaffected dog. As described above, PCR
the genotypes of an additional 88 dogs via was done with primers that encompassed
RFLP. SNPs were chosen based on p-value all 8 exons. Sanger sequencing was then
and availability of RFLP. performed, and the results were
Fifteen ng of purified DNA, specific compared between the two dogs and
20 µM forward and reverse primers against the published dog sequence.
(1.5uL), 300 µM dNTPs (1.5µL), 10x
buffer (1.5µL) in water (5.6µL), and TAQ Figure 2: RFLP for CFA19
polymerase (0. 5 µL) 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 Figure 2 Shows the 3 different band sizes after
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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 with
heterozygous AG and homozygous GG. For this a greater G allele frequency for affected
chromosome, G is strongly associated with dogs.
epilepsy; therefore, it is hypothesized that affected Conclusion
dogs have most often have a genotype of GG in the
studied position of 13,956,701bp.
Epilepsy in Australian Shepherds
is suspected to be an inherited disease
Results
and prior work has shown an association
between loci on CFA1 and CFA19 in this
Significant associations of SNPs
seizure disorder. By testing 23 affected
between affected and unaffected Aussies
and 65 unaffected Australian Shepherd
were still observed on CFA1 and CFA19
dogs for 2 different RFLP SNPs and
with the addition of (23) cases and (65)
comparing genotypes, the association of
controls, for a total of (42) cases and (86)
the loci on both CFA1 and CFA19 with
controls. The G allele in CFA19 (SNP -
epilepsy was confirmed. It is concluded
BICF2G63036912) is strongly associated
that there are likely genetic mutations in
with epilepsy and has a frequency of
both regions that contribute to the
47.3% in affected dogs. In CFA1 (SNP -
development of epilepsy in Australian
BICF2G630711334), the frequency for the
Shepherds.
C allele is also associated with epilepsy
Future work could involve WGA in
and has a frequency of 51.3% in affected
closely related breed such as Border
dogs.
Collies. Based on previous research on
The p-values for association for
Vizslas and English Springer Spaniels, the
these two SNP with epilepsy were 0.0103
lab team may continue their investigation
and 0.000750 for CFA1 and CFA19,
focusing on these 2 chromosomal areas. A
respectively. These results strongly
SNP Chi-square analysis could serve to
support our hypothesis of a causative
compare between breeds and determine
mutation near these locations in CFA1
if these associations are shared across
and CFA19. Affected dogs are more likely
breeds. In addition, new research may
to have G allele for the CFA19 SNP and the
involve looking at the other genes within
C allele for the CFA1 SNP as shown in
1-2 million base pairs near DOK6 to
Figure 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 case
versus the control Aussie selected.
Acknowledgements
I want to give special thanks to my
Figure: 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 Foundation
Figure 3.
• CFA1 showed statistical significance with a LSSURP
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5. RISE program R25GM059429-12
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epilepsy in four dog breeds BMC Genetics
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http://www.biomedcentral.com/1471-21
56/12/38
2. 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/10.11
11/j.1939-1676.2003.tb02455.x/abstract
3. Sutter N, Eberle M, Parker H et al.2004.
Extensive and breed-specific
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[Internet]. Available from:
http://www.ncbi.nlm.nih.gov/pmc/articl
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