Whole-genome Sequencing of non-prcd PRA in PWD

The Portuguese Water Dog Foundation will contribute $9,400 for Whole Genome Sequencing (WGS) on 4 different dogs [2 affected, 1 carriers, and 1 homozygous normal]. Samples will be tested at the WGS platform at the University of Bern, Switzerland. Dr. Aguirre’s eye lab is part of a consortium at Bern where they have WGS on 465 different dogs (at this point, none are PWDs). Bern will provide the initial filtering and data analysis, allowing Dr. Aguirre’s team to focus their analysis and to analyze the approximately 498 predicted genes in the interval in an expedited manner.

Although this proposal focuses strictly on non-prcd form of PRA, the results of the WGS studies will be critically important to other diseases of importance to PWD, for example microphthalmia/multiple congenital ocular anomalies (aka “puppy eye syndrome”) and possibly other diseases that rely on Genome Wide Association Studies (GWAS) to identify the genomic region of interest.

Any study using GWAS to map a disease interval will, much sooner than later, need high quality genomic sequence formation for the region. This comes from having access to as many different dogs/breeds as possible as well as having specific sequence information for the breed of interest from unaffected animals that can be used as disease controls. As the sequence information generated from this project will be publicly available, this resource will complement and inform research work being done on microphthalmia/multiple congenital ocular anomalies disorder as well as other diseases.

Research funded by the Portuguese Water Dog Foundation, Inc.


Gustavo Aguirre
School of Veterinary Medicine, University of Pennsylvania
Amount: $9,400

Genetic Background and the Angiogenic Phenotype in Cancer

This project will continue the researchers’ observations on gene appearance profiles in hemangiosarcoma from Golden Retrievers to German Shepherd Dogs and Portuguese Water Dogs, and it also will define how new targeted therapies may effectively control the disease in these and other dog breeds.


Certain dog breeds are prone to develop certain types of cancer; yet, there has been little progress to define genes or other factors that account for this risk. Our recent work on hemangiosarcoma was the first to demonstrate that a dog’s genetic background, defined by “breed,” can influence the profile of genes that are expressed by tumors. Among other important implications, this implies that certain breeds are diagnosed with specific cancers more frequently than others because of the behavior of tumors after they arise, and not simply because they arise more frequently. Specifically, this may apply to the observed predisposition for hemangiosarcoma seen in Golden Retrievers, German Shepherd Dogs, and Portuguese Water Dogs. Here, we continued to test this premise by evaluating genome-wide gene expression profiles in samples from dogs of various breeds.

Our results suggest that, while there are subtle differences that are influenced or modulated differently in tumors from dogs of different breeds, these differences may disappear when tumors are considered in their context as “tissues” that include microenvironment constituents. Rather, there appear to be distinct subtypes of hemangiosarcoma (perhaps with different biological behavior and prognosis?), which might arise from different cells of origin, or more likely, which develop in response to adaptation of the hemangiosarcoma cells to environments that show different patterns of inflammation, angiogenesis, coagulation, and hypoxia, each of which alters not only the predominant or favored differentiation of the tumor cells themselves, but also the way they instruct microenvironment cells to create a favorable niche.

This underscores the importance of looking at these tumors in their context as “new tissues” or “new growths” rather than at the cells in isolation as we work to develop more effective strategies for detection, diagnosis, and therapy. To follow on this premise, we evaluated new therapy approaches that target both tumor and microenvironment compartments. Specifically, one such approach also shows efficacy to kill tumor-initiating cells.

Data funded by this project grant and others allowed us to validate the therapy and move it to the clinic. Angiosarcoma Awareness, Inc. provided the initial funds to support a dose finding and efficacy trial where we will treat ~20 dogs with hemangiosarcoma using a bispecific ligand targeted toxin. We completed production of the molecule under “Good Manufacturing Practices” (i.e., suitable for use in human patients) and enrollment is ongoing. Finally, we identified other potential drugs to treat this disease – or perhaps more likely, the pathways they disrupt as potential targets for development of new therapies.

Co-sponsored with the AKC Canine Health Foundation, Grant Number: 01131


Jaime F. Modiano, VMD, PhD
University of Minnesota

Mechanistic Relationship of IL-8 in Cell Proliferation and Survival of Canine Hemangiosarcoma

Characterize the direct effects of IL-8 on HSA cells.


The hypothesis tested in this project was that interleukin-8 (IL-8) promotes growth and survival of hemangiosarcoma cells. This hypothesis was based on our previous results showing significant enrichment of IL-8 gene expression in hemangiosarcoma cells compared to normal endothelial cells isolated from non-malignant hematomas. Here, we confirmed that IL-8 is constitutively expressed by canine hemangiosarcoma cells in laboratory culture, as well as by primary tumors (fresh frozen samples). However, the levels of IL-8 are moderately variable among tumors.

Hemangiosarcoma cells in culture and primary hemangiosarcoma tumors also express IL-8 receptors (IL-8Rs). The receptors are expressed at comparable levels by virtually all the cultured cells and all the tumors, suggesting changes in expression of the receptor are unlikely to contribute to malignant behavior. We also confirmed that IL-8 binds to IL-8 receptors, and this interaction has functional consequences: IL-8 promotes signal transduction (calcium mobilization) in cultured HSA cells, and when we added IL-8 to cultured cells, they were able to “sense” this IL-8 excess and downregulated the expression of their own IL-8 gene. In contrast, if we blocked the interaction of their own secreted IL-8 with the receptor, they increased the amount of IL-8 gene expression. This is a classic response of compensatory regulation to negative feedback. Expression of a gene whose protein product turns on IL-8 gene expression followed the same pattern. It was downregulated when IL-8 was present in excess and induced when IL-8 was prevented from interacting with its receptor.

Despite its biological activity, IL-8 did not promote growth of hemangiosarcoma cells in culture, and IL-8 blockade did not hinder IL-8 growth in culture. When cells were deprived of nutrients and growth factors, they did not compensate by increasing production of IL-8; instead, IL-8 expression was reduced. And the addition of IL-8 did not prevent these nutrient-deprived cells from dying, and neither did it prevent cells treated with chemotherapeutic drugs from dying. Together, the data suggested that IL-8 did not directly mediate growth or survival of hemangiosarcoma cells in culture, refuting the initial hypothesis.

We then compared the gene expression profiles of cells and tumors that expressed high levels of IL-8 (and thus were adapted to growing in an environment rich in IL-8) with those of cells and tumors that expressed lower levels of IL-8 (adapted to growing in environments with relatively scant IL-8). The data show that cells adapted to high IL-8 environments had gene expression profiles indicative of greater inflammation, coagulation, fibrosis, and angiogenesis. These data suggested that IL-8 could be important to modulate the microenvironment and provide a suitable tumor niche. Experiments from an independent, complementary project funded by the National Canine Cancer Foundation showed that indeed, blocking IL-8 hindered the ability of hemangiosarcoma cells to establish a tumor niche in vivo. Finally, preliminary data suggest that IL-8 also may be necessary to maintain the tumor-initiating populations of canine hemangiosarcoma, by enhancing self-renewal. This hypothesis is under investigation in our newly funded project supported by AKC CHF.

Co-sponsored with the AKC Canine Health Foundation, Grant Number: 01429


Jaime F. Modiano, VMD, PhD
University of Minnesota

Genetic Analysis of Hypoandrenocorticism in Nova Scotia Duck Tolling Retrievers

Addison’s disease, also known as hypoadrenocorticism, is a deficiency of hormones that are produced by the adrenal glands and help regulate a dog’s metabolism, blood pressure, electrolyte balance and stress response. Though the disease is relatively uncommon in dogs, certain breeds—including Nova Scotia Duck Tolling Retrievers, Bearded Collies, Great Danes, Leonbergers, Portuguese Water Dogs, Standard Poodles and West Highland White Terriers—have a much higher risk than the general dog population.


Researchers identified a region of the genome that is associated with the development of Addison’s disease in Nova Scotia duck tolling retrievers. Additionally, it appears that dogs that are homozygous (both chromosomes carrying the same genes) with respect to this region are at greater risk of developing Addison’s disease, even at a young age (under 2 years). Although additional genes are likely involved, this information is the first step toward understanding the genetics of this disease and developing a genetic test that will help eliminate Addison’s disease through informed breeding practices. This fellowship training grant also provided hands-on training for a veterinarian who is pursuing a research career.

Co-sponsored with the Morris Animal Foundation, Grant Number: D08CA-402


Angela M. Hughes, DVM
University of California at Davis

Pooled Association Mapping for Canine Hereditary Disorders

To develop a system that will map genetic traits causing health problems.

More than 450 canine genetic traits are listed on the Online Mendelian Inheritance in Animals list. These traits affect all body systems in dogs and can cause health problems ranging from mild disease susceptibility to severe illness and death. Researchers will use a genetic tool called the Affymetrix Canine SNP Chip to develop a mapping strategy using pooled DMA samples that will map genes for hereditary canine disorders. This method promises to be 10 times more efficient and cost effective than current methods for analyzing canine inherited traits. Once this method is validated, it will be used to map a series of hereditary retinal disorders affecting multiple breeds.

Co-sponsored with the Morris Animal Foundation, Grant Number: D07CA-085


Gregory M. Acland, BVSc
Cornell University

Dr. Acland is a professor of medical genetics at Cornell University and an adjunct p of ophthalmology at the University of Pennsylvania. He received his veterinary degree from the University and completed a residency and postdoctoral work at the University of Pennsylvania.

Reciprocal Relationship of PTEN and p21 in Canine Cancer

An estimated one out of every two dogs alive today will get cancer in its lifetime, and as many as 50 percent of those will die from the disease. Despite significant gains in cancer treatment, a thorough understanding of why cancers arise and why they behave as they do is essential to improving prevention and treatment. For this project, researchers will investigate two proteins whose interactions appear to be intimately tied to the behavior of two serious cancers, melanoma and hemangiosarcoma. What they learn may help to test targeted therapies for these cancers and significantly improve the lives of affected dogs.


Cancer is the leading cause of disease-related death in dogs, which is why the Morris Animal Foundation and the Portuguese Water Dog Foundation have invested considerable resources to understand this group of diseases to develop more effective treatments. Cancer happens when genes that control the balance of division and survival cease to function normally in a cell and cause it to become malignant. One of these genes is called PTEN. The protein product of this gene generally restrains cell division, in part by controlling another protein called p21.

Scientists at the University of Minnesota and at the University of California, Davis have found that using compounds to lower the levels of p21 in some tumors decreased the resistance to conventional chemotherapy drugs. They also concluded that chemotherapy resistance is sometimes unrelated to abnormalities of PTEN, while it is often associated with elevations of p21. The results have allowed the investigators to justify efforts to move these compounds to the next step of clinical development.

Co-sponsored with the Morris Animal Foundation, Grant Number: D06CA-065


Jaime F. Modiano, VMD, PhD
University of Minnesota

Mapping Genes Associated with Canine Hemangiosarcoma

Hemangiosarcoma (HSA), a malignant tumor of vascular endothelial cells, is a significant health concern in dogs, with an incidence of ~2% of all tumors. A national health survey of Golden Retriever reported that neoplasia accounted for >60% of all reported deaths and HAS was the most common malignant tumor affecting >15% of Golden Retrievers. A particularly high disease incidence of hemangiosarcoma in Golden Retriever suggests a genetic susceptibility.

The purpose of this study is to identify the mutations causing the increased risk for hemangiosarcoma in Golden Retriever. To do this, we have proposed to compare the genotypes of dogs diagnosed with HSA with healthy older dogs using a statistical analysis.


To date, we have collected 125 blood samples from Golden Retrievers diagnosed with HSA and more than 400 healthy Golden Retrievers over 8 years old. We have identified six regions in chromosomes associated with HSA and are have narrowed these to precise regions (a few hundred thousand base). We now need to find the precise mutations that cause the disease and link them to functionality. In the long term, this work should allow the development of specific genetic tests for carriers of HSA. Ultimately, understanding of the disease biology, which will lead to identification of target genes for prevention, early detection and novel treatments of this malignancy.

Co-sponsored with the AKC Canine Health Foundation, Grant Number: 00593A


Kerstin Lindblad-Toh, PhD
Broad Institute at MA

Identifying Genes Regulating Addison’s Disease in the Portuguese Water Dog

Addison’s disease, or primary adrenocortical insufficiency, is characterized by destruction of the adrenal cortex, resulting in the inability to produce cortisone when stimulated with the hormone ACTH. In Portuguese Water Dogs (PWDs), this disease occurs with a frequency of 1-2 percent, and is a heritable autoimmune disease of low penetrance, caused by several interacting genes.

Using both new and existing data, we propose to identify regions of the PWD genome that contain genes regulating the frequency of Addison’s disease. Within those large regions we propose to identify the specific DNA sequence variants that are associated with Addison’s.


To date we have obtained DNA from about 90 Addisonian PWDs, as well as a number of unaffected PWDs, for which no family history of Addison’s is reported. We have already identified two genomic regions, on canine chromosomes 12 and 37, that appear to be associated with the disease. To identify candidate genes, we will make selections using the newly available canine genome sequence, as well as the more detailed human genome sequence. Once affected gene disease frequency is identified, our long term hope is that prognostic tests can be developed that will aid breeders in selecting the most genetically compatible dogs for future.

Co-sponsored with the AKC Canine Health Foundation, Grant Number: 00589B


Elaine Ostrander, PhD
National Human Genome Research Institute

Mapping Refinement of Quantitative Trait Loci for Canine Hip Dysplasia

Hip dysplasia is one of the most common inherited traits in dogs with an extremely high incidence in some large breeds. It is caused by mutations in multiple genes. In previous studies, investigators discovered the genetic markers that point to the chromosomal regions that harbor the genes that contribute to hip dysplasia.

In this study, they will narrow down these regions through additional genetic evaluation. By narrowing the regions that harbor hip dysplasia genes across breeds, they hope to discover the contributing mutations and use that information to design genetic tests that can be used to prevent the propagation of dysplastic dogs.


Researchers identified the first mutation associated with canine hip dysplasia in Labrador retrievers. This mutation could be used in conjunction with a panel of genetic markers to identify susceptible dogs. The team also learned that there is no single gene identified for hip dysplasia in dogs. This study pointed to several other genes that contribute to the disease, and researchers believe they will discover these genes through the process they have developed.

Based on their findings, the researchers hypothesize that identifying dogs that are resistant to hip dysplasia will involve a panel of genetic markers that may be breed specific. Breeders could then use these panels in conjunction with breeding values to genetically screen puppies before the mutations themselves can be identified. This project lead to the discovery of the first gene associated with canine hip dysplasia.

Co-sponsored with the Morris Animal Foundation, Grant Number: D04CA-135


Rory J. Todhunter, BVSc, PhD
Cornell University

Gene Expression Profiling of Relapsed Lymphoma in Dogs

Lymphoma is one of the most common cancers in dogs. Breeds with a high incidence include Boxers, Golden Retrievers, and German Shepherds. It is also one of the most treatable cancers – more than 90 percent of dogs treated with chemotherapy go into remission. Unfortunately, most dogs relapse and eventually die from the disease.


The investigators compared gene-expression patterns of tumors before and after chemotherapy to determine changes that took place in the dogs that relapsed. They identified important candidate genes and different subtypes of lymphoma that will help in the development of new cancer treatments. These candidate genes must now be independently validated in another set of patient samples but have already contributed greatly to the understanding of genes associated with relapse and potential new therapeutic targets.

Co-sponsored with the Morris Animal Foundation, Grant Number: D03CA-132


W. C. Kisseberth, DVM, PhD
Dip. ACVIM (Oncology)
Ohio State University