RESEARCH POSTER PRESENTATION DESIGN © 2012 www.PosterPresentation s.com Ventricular septal defects (VSDs) are the most common congenital heart defects reported in horses. VSDs are characterized by an abnormal communication between the left and right ventricles that varies in size, location, and clinical relevance. Even small, high velocity defects known as restrictive VSDs can lead to impaired cardiac function if the cusps of the aortic valve are drawn into the defect, resulting in aortic insufficiency. When severe, these lesions result in volume overload in the left heart and ultimately congestive heart failure. Arabian horses are overrepresented for incidence of congenital heart defects including VSDs, and a genetic basis for VSDs in the breed is considered likely. Identification of genetic markers associated with VSDs would facilitate the isolation of a VSD causative mutation and ultimately enable the development of a genetic test for Arabian breeding managers to promote the health of the breed and limit the prevalence of this defect. Introduction Cardiac auscultation and echocardiographic evaluations were used to phenotype 13 affected and 38 normal Arabian horses. DNA was extracted from whole blood samples and genotyped using the Axiom® Equine Genotyping Array that contains 670,000 SNPs across the equine genome. Materials and Methods Ventricular septal defects (VSDs) in Arabian horses are associated with a single genetic locus identifiable through a genome-wide association study (GWAS). Hypothesis Figure 3. Manhattan plot illustrating the most significant SNP association on chromosome 25 in the EMMAX analysis. Bonferroni adjusted and raw P values are displayed. Results Discussion Despite the suspected heritability of VSDs in Arabian horses, no prior studies have undertaken to identify SNPs, haplotypes, and genes associated with VSDs in this breed. Case vs. control GWAS was successful in identifying a chromosomal region of interest. Chromosome 25 has the strongest association with VSDs and withstood Bonferroni correction. Phenotypic diversity is a well-known feature of VSDs in many species. A specific form of VSD as a component of a more severe congenital malformation known as tetralogy of Fallot is rarely diagnosed in Arabian horses and other horse breeds. Our data suggests that tetralogy of Fallot is genotypically distinct from the VSD variety tested in this study (perimembranous). Further defining the identified region of interest and investigating variants in candidate genes through whole genome sequencing will ideally guide future translational research and aid in the understanding of VSD pathogenesis. References 1. Leroux, A. A., et al. "Prevalence and Risk Factors for Cardiac Diseases in a Hospital-‐-Based Population of 3,434 Horses (1994–2011)." Journal of Veterinary Internal Medicine 27.6 (2013): 1563-1570. 2. Hall, T. L., K. G. Magdesian, and M. D. Kittleson. "Congenital cardiac defects in neonatal foals: 18 cases (1992–2007)." Journal of veterinary internal medicine 24.1 (2010): 206-212. 3. Thomas, Bill. "The Equine Heart." CEH Horse Report: A Publication of the Center for Equine Health, UC Davis School of Veterinary Medicine 24.4 (2006): 1-12. Center for Equine Health. UC Davis School of Veterinary Medicine. Web. 10 Jan. 2015. <http://www.vetmed.ucdavis.edu/ceh/>. 4. Kahn, Cynthia M. "Congenital and Inherited Disorders of the Cardiovascular System in Horses." The Merck/Merial Manual for Pet Health. Home ed. Whitehouse Station, NJ: Merck, 2007. Print. 5. Norton, Elaine. "Genome Wide Association Study for Equine Exertional Rhabdomyolysis in Standardbred Horses." Plant and Animal Genome XXII Conference. Plant and Animal Genome, 2014. Acknowledgements Department of Medicine & Epidemiology, School of Veterinary Medicine, University of California Davis E Morgan, E Ontiveros, K Estell, J Stern Identification of Genetic Markers for Ventricular Septal Defects in Arabian Horses Location Unadjusted P Bonferroni P Chr25.18647558 4.14E-011 1.30E-05 Chr25.18658851 4.70E-09 0.0015 Chr25.18659166 4.70E-09 0.0015 Mixed linear model analysis (EMMAX) was performed to further reduce the effects of population stratification. QQ plot improved: results displayed. Chromosome 25 significance persisted after 330,000 permutations. Figure 1. A 2D and color Doppler echocardiogram still image is provided of a 2 year old Arabian gelding. The image is obtained from the right thorax. The VSD (arrow) is visualized with turbulent flow passing into the right ventricle, just below the aortic valve (asterisk). Table 1. The SNPs most strongly associated with VSDs from the EMMAX analysis are detailed by location in EquCab2 and test statistics. Figure 4. Additive association model with 330,000 permutations. The pink line denotes a genome-wide significance value of P=0.05. Phenotype Auscultation: A VSD is associated with two possible heart murmurs. The shunt itself causes a loud, holosytolic murmur with a point of maximal intensity over the right thorax. A murmur of relative pulmonic stenosis is often auscultated as well. Though the right ventricular outflow tract may be anatomically normal, the increased volume of blood leaving the right ventricle results in a holosystolic crescendo- decrescendo murmur most audible over the pulmonary valve region on the left thorax. Echocardiogram: Echocardiography is used to definitively diagnose a VSD and classify its location. With cardiac ultrasound, the defect is visualized as a communication between the two ventricles. The velocity and direction of flow through the shunt is measured to determine if the defect is restrictive (hemodynamically insignificant) or non-restrictive. The downstream effects of volume overload, such as dilation of the pulmonary artery and left atrium and eccentric hypertrophy of the left ventricle, are assessed. This study targets perimembranous VSDs. Extracted DNA from whole EDTA blood and formalin-fixed, paraffin embedded tissue Hybridized DNA of 12 cases and 39 controls to Axiom® Equine Genotyping Arrays Conducted Genome- Wide Association Study (GWAS) and quality control using Golden Helix Software 37,414 unmapped SNPs were excluded for analysis Quality control removed an additional 257,795 SNPs (minor allele frequency inclusion threshold of 0.08, maximum per individual missing genotype rate of 5%, and maximum Hardy Weinberg Equilibrium P-value of 0.001) 375,587 SNPS were retained and analyzed after quality control and exclusion of unmapped SNPs. 5 cases and 8 controls were removed based on MDS plot indicating population stratification. Case vs. control association with 8 cases and 30 controls yielded a genome inflation factor of 1.28 and identified a tightly clustered region of association on Chromosome 25. The top SNP identified on Chromosome 25 yielded a Bonferroni P value of 1.30E-05 Figure 5. QQ Plot demonstrating that population stratification has been corrected after adjustment by MDS and EMMAX. Figure 7. Healthy 2 month old Arabian colt. Figure 4. QQ Plot prior to adjustment for population stratification (MDS and EMMAX; Genome inflation factor=3.43). (Praw=4.14E-011) (Padj=1.30E-05) Figure 2. Spectral Doppler echocardiographic image depicts blood flow across the VSD. The flow measures 5.5 m/sec toward the transducer in systole. This confirms the shunt direction is left to right and the velocity confirms that this VSD is hemodynamically insignificant (restrictive). P=0.026 Figure 6. Haplotype analysis. Complex VSD represents tetralogy of Fallot- a genetically distinct disease complex in humans.