January 2023 Cataract Research Update
University of Pennsylvania,
Personnel:University of Pennsylvania
Gustavo Aguirre, VMD, PhD; Principal Investigator, Leonardo Murgiano, PhD; co-Principal Investigator, Anil Sigdel, PhD; Postdoctoral Researcher, Jessica Niggel, M.Sc.; Research Scientist
In this installment of the American Cocker Spaniel (ACS) Inherited Cataract study report, we would like to again thank all the ACS breeders and owners for their continued support and participation of our study. Compared to the previous report, the number of dogs participating in the study increased from 995 to 1005 – the last annual increment to date.
In our pursuit for the detection of the causative variant(s) for cataract in ACS, we encountered issues and ‘roadblocks’ that initially slowed progress during the tenure of the grant years. These were due to the very likely complex inheritance of the disease or of the low informativeness of the SNP data in light of the current population of cases and controls available; additionally, we found that the standard SNP-chip markers were of low informativeness. For this reason, we opted for a radical approach putting whole genome sequencing (WGS) as a priority and this approach has recently brought very significant results.
We sequenced a total of 12 early onset cases. As a first step of our analysis, we focused on small SNPs and variants, which are easily genotyped as markers. Once these were detected, these results were filtered against two different datasets – the canine WGS files generated by our lab, and against a recently accessible WGS dataset comprised of more than one thousand dogs. This new influx of controls helped us filter out a large amount of distracting variants and factors that would normally hamper the search for associated genetic mutations. The cases were added as we went, analyzing the preliminary output and then evaluating if more were required, and this was done on an iterative manner. The new reduced costs of WGS allowed us to implement this strategy.
Of note, we previously stated that our plan consisted of employing an alternative WGS method (low pass WGS) that is sufficiently accurate for our purposes but has a significant decrease in its cost. We stated we would genotype 10-15 American cocker cataract cases and controls (each) and integrate such data to the dogs already sequenced. The plan was supposed to consist in using this dataset primarily for a GWAS that uses millions more markers than its standard version. While this is correct, preliminary evaluation of similar datasets in our possession put the variant detection reliability in question for the type of analysis planned. Additionally, we acknowledged the availability of new canine references and datasets, therefore changing our plan to a more direct and pragmatic sequencing of the best cases.
Genome-Wide Association Study and genotyping, nonetheless continued. As reported, most significant population sub-division is among those cataracts appearing in the 2-5 years of age range and progressing, as well as the ones with a later (5-8 years of age) onset and progression. We still use the data generated in this way to detect general trends and potential regions (three of these were considered the most relevant statistically).
After a first round of WGS of early onset cases, we analyzed the variants detected, we filtered them, and we grouped them by impact and gene function. This led us to carry out a (very recent, Nov 2022) submission of additional early onset cases. We can say that this very straightforward approach paid off extremely well!
In fact, after filtering against two different databases, we remained with variants in the order of tens (which is, for a whole genome of millions of variants, extremely low). Of these, interpolating the WGS with the mapping info, three are the most promising. Two of them fall within one of the candidate regions identified by the GWAS analysis shown in the poster attached to this report. The poster was presented at the 2022 International Conference on Canine and Feline Genetics and Genomics conference in Huntsville, Alabama, and the American Cocker Spaniel Foundation was acknowledged as the main supporter of the work. Our next steps will consist in Sanger sequencing the whole sample pool and observe the segregation to point out a viable marker. After this, we will:
- Carry out a similar pipeline for the same dataset but checking for larger variants, which require a more complex pipeline to detect and filter, but we cannot exclude to have any regulatory role and must therefore be checked.
- Repeat the whole above pipeline, included point (1) for later-onset cataract ACS dogs. In fact, we have identified other 12 cases suitable for this high-throughput process which we expect will lead to similar exciting results. As an additional measure, once the critical number of dogs have been sequenced, we will search for any putative regulatory region that could have a role in determining the age of onset.
On the breeders’ part, we once again stress the critical importance of updates and new samples. Lack of updates endangers the quality of the dataset and therefore the results. We also ask any owner of non-affected, older dogs to send samples for the study. We think that due to the nature and inheritance of the condition, non-affected controls are vital. Positive feedback and large numbers are the keys to success. Now, our focus will be on the Sanger sequencing of the early onset data and on the application of the same strategy for the detection of causative markers for the later onset cataract in ACS.
The Foundation Cataract Study was presented at the 2022 International Conference on Canine and Feline Genetics and Genomics Conference by Leonardo Murgiano. The graphic used for the presentation follows and outlines the study and the results to date.
July 2022 Research Update
For many decades now, the American Spaniel Club Foundation remains the only nonprofit 501c3 with the mission of bettering the life of Cocker Spaniels through research, education, and rescue. To achieve this mission, we focus on three initiatives.
- We contribute to and/or directly sponsor veterinary research programs to improve the health of Cocker Spaniels by understanding and attempting to eliminate the diseases, genetic abnormalities, and injuries that impact them.
- We work to ensure that every Cocker Spaniel has a good home and adequate veterinary care by providing financial assistance to and promoting a national network of affiliated local rescue organizations. This effort has within the last year been reinvigorated under the leadership of Rescue Chair Andrea Floyd.
- We attempt to provide education and information on health and research topics that promote intelligent breeding, care, and knowledge of Cocker Spaniels. Our strategy in the future is to expand this effort through a network of noted research scientists and canine experts using recent legacy-giving contributions.
These three pillars are funded through frequent fundraising programs and legacy giving initiatives through the generosity of American Spaniel Club members, the Cocker Spaniel fancy in general, and some corporations. This year the Foundation was most honored to receive a major legacy memorial bequest that has given us the funding to think more strategically in supporting our three initiatives, particularly on the research front. We hope this legacy giving will continue in the future. We are launching a reinvigorated Legacy Giving drive at the July National through the leadership of Vice President Lindy Bennett.
For this specific Foundation communication that you are reading now, we would like to focus on the current and future health and research direction of the Foundation. Primary in our efforts is the continued support of the cataract research led by Dr. Aguirre and his research team at the University of Pennsylvania. We expect to release some positive updates on the annual mid-year webinar later in August with Dr. Aguirre and his newly expanded team and we will share them via social media and on our website as soon as available. As always Dr. Aguirre reminds everyone of the continued need for both affected and clear cases across all age groups as well as the annual exams and updates on the clear dogs already in the study.
We are also currently sponsoring with the AKC Canine Health Foundation four research studies on diseases that have a serious impact on Cockers. They are listed below. More information is available on our website at ASC-F.Org. If you have any further questions on this research, contact us at AsktheFoundation@gmail.com or see Health and Research Chair, Fulvia Bartoli if you are attending the ASC National in July.
- OX40 Checkpoint Molecule Targeted Antibodies for Cancer Immunotherapy in Dogs The Principal Investigator is Dr. Steven Dow Colorado, State University.
- Genetics of Primary Angle Closure Glaucoma in American Cocker Spaniels Principal Investigator: Dr. Sara Thomasy, DVM, Ph.D. University of California, Davis. We will be hosting Dr. Thomasy on a webinar in the future where she will talk more about her research and glaucoma in Cockers.
- Plasminogen-activator Inhibitor-1 (PAI-1) and Impaired Fibrinolysis in Immune-Mediated Hemolytic Anemia. Principal investigators Dr. Tracy Stokol at Cornell University and Dr. Steven Friedenberg, at the University of Minnesota.
- Clinical Trial for Evaluation of Propanolol and Doxorubicin in the Treatment of Canine Hemangiosarcoma. The Principal Investigators are Dr. Erick Dickerson and Dr. Antonela Borgatti at the Univ of Minnesota. The research is also carried out with the Co-Investigators at the Univ. Of Purdue and Univ of Pennsylvania.
One of the outcomes of our strategic planning last year was to study and commit to and/or create specific research objectives and fund annual program budgets to support the rather lengthy amount of time most research requires. We have been fortunate to have the opportunity to directly fund, through many contributions now over $400,000, the Cataract Research Study with the University of PA led by Dr. Aguirre. Most of the research we fund is through the AKC CHF and will benefit Cockers but very few are Cocker specific. The question for the Foundation Board was what research should be a target along with our current commitments? We have taken two approaches to answer this.
- First, we realized that since one in 2004, the Foundation has not conducted a health survey within our fancy 2004 on what are the disease and health concerns of breeders and owners. The results of such a survey would be vital in directing where the Foundation should focus our search for creating and funding new programs or supporting existing ones. Our Health and Research chair Fulvia Bartoli along with her committee is working hard to get a confidential and secure health survey out as soon as possible. We ask for everyone’s participation when that occurs. If you have any input or specific concerns on what we should be surveying please contact Fulvia or any other member of the Foundation Board.
- Second, we reached out to the Foundations of the other spaniel breeds and through the leadership of Health and Research Chair Fulvia Bartoli, we are excited that we have formed a discussion group called the Spaniel Health Initiative with the intent to share health and research focus, programs, and funding across the clubs. We hope to find commonality among the spaniel breeds where we can leverage and fund research together as a group. Included in this group, at the moment, are the English Cocker Spaniel, English Springer Spaniel, Welch Springer Spaniel, and Clumber Spaniel national breed clubs. This exciting new Spaniel Health Initiative has met now three times and the main takeaways and strategies are the need to update our own Health Survey, identify the common causes affecting the Spaniel breeds, and share participation in clinical trials in the future. More to come.
As the Foundation expands the support of research programs in other disease areas in the future, we want to emphasize that your participation in the studies or clinical trials is crucial to their successand is as important if not more so than the monetary support. So please expect our pleading in this area to continue and thank you in advance for that support.
All of these initiatives are to better the lives of Cocker Spaniels everywhere. Thank you for your attention to this long communication. If you have questions or comments, please utilize our Foundation email at AsktheFoundation@gmail.com or talk to any of our Directors via email or at a show. As Foundation President and Executive Director, and on behalf of the Board, I want to thank all of you that have generously given your money, time, talent, and canine DNA in the past. We look forward to your support in the future.