2013 Research Funded by YTCAF
Each year the AKC Canine Health Foundation send the YTCA Foundation a list of grant requests for funding that they have screened and think would be of interest to breeders and owners of Yorkshire Terriers as well as dog fanciers in general. As an independent entity spun off from the Parent club to preserve our tax exempt status, the YTCAF receives no financial support from the YTCA's participating in efforts such as the Purina Circles program. Instead all of our funding comes from memorials made by individual donors and fund raising efforts such as the online auctions and breeding calendar sales.
The Board members carefully considered the following factors in each request in order to optimize research dollars:
Potential of the research to benefit Yorkshire Terriers in particular and all dogs in general.
Previous research success of the primary investigators
Use of the grant project to help veterinary students, new veterinarians and academicians get started in their chosen field of research (Acorn projects).
The Board voted to support the following CHF grants for 2013.
O1918-G: Discovery of Novel Protein, Blood, and Epigenetic Biomarkers of Lymphoma Risk, Classification, and Prognosis in Golden Retrievers
Jeffery N. Bryan, DVM, University of Missouri, Columbia
Lymphoma strikes 1 in 8 Golden Retrievers, approximately one-third of the cases being B cell. While T cell classifications currently inform therapy choices for dogs, B cell classifications have been investigated little in Golden Retrievers. Epigenetic DNA methylation changes clearly underlie lymphomagenesis in humans, but have been evaluated minimally in dogs. Cancers contain tumor initiating cell (TIC) populations that resist therapy by expressing efflux pump and pro-survival genes that have not been identified clinically in lymphoma of dogs. We propose to improve diagnostic, classification and prognostic ability using flow cytometry paired with biopsy to characterize the B cell lymphomas of Golden Retrievers. With these same samples, we will identify DNA methylation changes in lymphoma cells not present in normal cells to develop biomarkers of each class of lymphoma and identify new therapy targets for affected Goldens. More significantly, because DNA methylation changes occur so early in the process of cancer formation, we hypothesize that they could serve as biomarkers of risk, allowing medicine or diet to prevent lymphoma in Goldens before it develops. Finally, we propose to identify TIC in lymphoma biopsies to characterize stem-like cells by surface markers and DNA methylation changes. Identifying these cells will aid therapeutic strategy development. Eash project advances a current frontier of research. By performing them in parallel, the markers from each can be combined, correlated, and translated into biomarkers of risk, diagnosis and prognosis to advance the prevention and management of lymphoma in Golden Retrievers.
Progress continues at all 3 institutions. The proposed immunohistochemical evaluations and flow cytometry techniques are developed and are being refined as cases are added to the series. DNA from purified neoplastic B cell samples from Golden Retriever lymphomas from CSU has been processed and prepared for sequencing. Preliminary sequencing experiments have shown that the available material is sufficient to generate reliable methylome sequence. TAMU has successfully generated tumor initiating cell populations from cultured lymphocytes and is optimizing the procedures to be performed on fine-needle aspirates of lymphoma nodes. It appears that multiple aspirates will be necessary to get all the material needed for characterization. Sufficient TIC cells can be generated for sequencing with the protocol in place of MU. The personnel for the studies are now in place.
01889-G: Developing Markers to Diagnose and Guide Cancer Treatment in Golden Retrievers Based on Newly Discovered Heritable and Acquired Mutations
Jaime F. Modiano, VMD, PhD, University of Minnesota
Lymphoma and hemangiosarcoma are major health problems in Golden Retrievers, causing both suffering and premature death. As part of our ongoing project, Discovery and Characterization of Heritable and Somatic Cancer Mutations in Golden Retrievers, we have identified several regions of the genome that contain genetic somatic mutations in tumors that occur recurrently in both cancers, some of which are linked to duration of remission when treated with standard of care. Our results indicate that a few heritable genetic risk factors account for as much as 50 % of the risk for these cancers. These findings offer the potential to develop tests and strategies for DNA tests that can predict risk for individual dogs, as well as to manage risk across the population as a whole. Indeed, both the inherited risk factors and tumor mutations point to pathways that have been implicated in the pathogenesis of LSA and HAS and thus should inform the development of targeted therapies. In this proposal we aim to find the precise mutations for the heritable genetic risk factors and to validate markers (mutations) used to determine risk at the heritable loci in a larger independent population of Golden Retrievers from the USA and from Europe in order to develop robust risk prediction tools and an accompanying DNA test. We will identify and characterize tumor mutations and study their relationship to the heritable risk factors, tumor pathogenetic mechanisms, and disease outcome.
This project has completed the first six months. For analysis of the DNA sequence mutations we identified previously, whole genome sequencing of DNA from several golden retriever blood samples from dogs affected by lymphoma and hemangiosarcoma has been performed. A large number of DNA sequence variants has been detected and are being carefully analyzed. Additional DNA sequencing is underway to provide more information. Collection of blood samples from golden retrievers across the US and Europe has been ongoing and over the coming months we will assess the frequency of the risk haplotypes within these two populations.
For evaluation of DNA sequence changes in lymphoma and hemangiosarcoma tumor samples we are compiling cases that will provide sufficient material for multiple analytical approaches. These approaches primarily include an assessment of genome wide DNA copy number changes in the cancer cells, and a direct comparison of the DNA sequences of all the coding regions of the genome in DNA isolated from blood and tumor cell populations. These data will identify which genome regions and which genes are associated with the diseases.
Functional correlation of genotypes and tumor phenotypes/outcomes will be feasible from TMA data. Samples from dogs with and without the germ line risk haplotypes are included in the TMAs, and virtually all the remaining samples have paired blood for extraction of germ line DNA and genotyping. The data indicate there is heterogeneity in signaling pathways and inflammation, suggesting the correlation analyses will be informative. These data will improve our ability to select samples for the final objective.
01731: Potential Association Between Altered Gut Microbiota and Development of Meningoencephalomyelitis of Unknown Etiology (MUE) in Dogs
Nicholas Jeffery, BVSc, PhD, Iowa State University
This Project is aimed at finding a cause of ‘meningoencephalo-myelitis of unknown etiology’, otherwise known as ‘MUE’, which is a summary term for diseases such as ‘GME’. This group of conditions cause serious neurologic disease in dogs, especially small breeds, including blindness, loss of balance, seizures and paralysis – but the cause is currently unknown. A recent experimental breakthrough has incriminated bacteria in the digestive system as triggers for a similar disease in laboratory mice and rats. The main purpose of this project is to find out whether imbalances in the number or type of digestive system bacteria might also be a cause for MUE in dogs. If it was to be true, it would open a whole new avenue of approach to treatment of affected dogs and might also produce information useful for treating neurologic disease in humans. To carry out this investigation we will analyze fecal samples collected from affected dogs for their content of bacteria. We will then compare the numbers and type of bacteria with those we find in fecal samples from unaffected dogs. Because we will be examining many dogs with MUE we will also be able to ask owners other questions about their dogs’ recent medications and medical history to determine whether there are other risk factors that might be associated with MUE.
This project is progressing as we were hoping that it would. We have collected questionnaires and fecal samples from 22 affected dogs so far, out of an aimed-for total of 30, plus 12 of the aimed-for total of 30 control dogs. We cannot analyze the data until it is all collected; the current phase is simply collecting the material for analysis at a later date.
This project is progressing as we were hoping that it would. We have collected questionnaires and fecal samples from 7 dogs so far, out of an aimed-for total of 30. We cannot analyze the data until it is all collected; the current phase is simply collecting the materials for analysis at a later date.
Marcello Pasquale Riggio, PhD, University of Glasgow
Our knowledge of the causes of gum disease in dogs is poor, even though this is one of the most common diseases of dogs and causes severe pain and tooth loss. However, it is widely thought that certain bacteria may cause the disease, as is the case in the human form. Most of the research carried out so far into the causes of gum disease in dogs has focused on growing specific bacteria from clinical samples. However, since many types of bacteria in the mouth cannot be grown in the laboratory, we have an incomplete understanding of the bacteria linked to health and disease of the mouth. To over come this problem, we shall use the most modern, curring edge laboratory technology available (known as ‘high-throughput sequencing’) to provide an in-depth understanding of the types of bacteria that cause gum disease in some dogs but not others. The method detects the DNA of bacteria rather than live bacteria and allows bacteria to be identified and quantitated without the need to grow them from clinical samples. The biggest advantage of this method is that, as well as detecting known tryp0es of bacteria, we can also identify bacteria that cannot be grown in the laboratory as well as new types which have not been discovered previously and which may help to cause the disease. This study will give us the most up to date knowledge on gum disease in dogs and will help in the development of vaccines and improved treatment methods for this disease.
This study will identify the bacteria that cause periodontal disease (gum disease) in dogs of all ages and breeds, and covers laboratory costs. In April 2013 we appointed part-time research technician to assist with the laboratory work and data analysis for the study. We have collected all samples required for the study. Clincians collected a plaque sample from the gums of 20 dogs with a healthy mouth and plaque from 20 dogs with an advanced form of periodontal disease (periodontitis). Since the required numbers of samples have now been collected, we are currently identifying the types of bacteria present using the modern laboratory method known as "high-throughput sequencing." The results should be available by July 2013. This will greatly increase our knowledge of the type of bacteria that cause gum disease in dogs, with a view to developing improved treatment and prevention methods in the future.