Photo by Louise Laing
John Dillberger, DVM, Ph.D., & Miranda Levin, ONB
Reprinted from the January/February 2021 issue of The Claymore.
Like everything else this year, our research was impacted by COVID. Many projects were delayed, but we did manage to make some progress on a few fronts.
Report by John Dillberger
As you may remember, the SDCA is working with a research group headed by Dr. Carlos Alvarez, of Nationwide Children’s Hospital and The Ohio State University, to identify genetic risk factors for osteosarcoma in Deerhounds. Not surprisingly, Dr. Alvarez’s laboratory was closed most of 2020 due to the COVID-19 pandemic. The lab began to reopen in November and plans to be back to normal operation in early 2021.
Despite the closure, the Alvarez group made progress, which he shared with us in an annual update in December. I have rearranged the update into sections and translated some of the scientific terms to make reading easier for folks without a background in genetics. For those who want the bottom line, here are the take-home messages:
A new look at old data: 2020 Update on Osteosarcoma in Deerhounds
In 2013, Elinor Karlsson and her co-workers published a paper describing 33 genetic variants associated with osteosarcoma in Greyhounds, Irish Wolfhounds, and/or Rottweilers. In 2019, Dr. Isain Zapata in the Alvarez group published a paper identifying a 34th osteosarcoma-related genetic variant in Greyhounds, Irish Wolfhounds, and Rottweilers.
In 2019, the Alvarez group used two analysis methods to evaluate all 34 osteosarcoma-related variants across the three breeds to estimate how much each affected overall risk. For those who are curious, one analysis method is called LASSO, which stands for Least Absolute Shrinkage and Selection Operator.
Based on their analyses, the Alvarez group divided the 34 osteosarcoma-related variants into four tiers, ranked from greatest contribution (Tier 1) to lowest contribution (Tier 4). For 13 of the 34 variants, they were able to nominate the likely gene involved. Importantly, the group then tested their risk modeling formulas in a separate cohort of Greyhounds. The risk model predicted quite accurately whether or not a Greyhound had osteosarcoma based on its set of genetic variants, giving confidence in the method.
Dr. Alvarez’s group then turned to Scottish Deerhounds and asked the following question:
How often is each of the 34 genetic variants associated with osteosarcoma in other breeds present in Deerhounds with osteosarcoma?
To answer this question, Dr. Alvarez’s group used genome-wide genetic marker data from 10 Deerhounds that were published (along with data from 160 other breeds) by Dr. Elaine Ostrander’s group in 2017. Those dogs were collected as a random sample of the Deerhound population, without regard to osteosarcoma (that sample collection preceded current collaborations of the SDCA). At least one of those dogs went on to develop osteosarcoma, but the status is unknown for the others. The genetic data were in the form of SNPs (single nucleotide polymorphisms). Because each Deerhound has two sets of chromosomes, one from its dam and one from its sire, the data set consisted of SNP maps of 20 sets of chromosomes from a random sample of Deerhounds.
Dr. Alvarez’s group then analyzed the Deerhound data for each of the 34 osteosarcoma-related genetic variants. Although the data set was small, it was big enough to identify any variant that was not rare because geneticists define a rare variant as one that is present in less than 5% of a population (that is, which is present in fewer than one in 20 chromosomes). By this criterion, any variant that was present in the Deerhound data set was not rare.
Of the 34 osteosarcoma-related variants, 3 were not present in any of the chromosome sets from Deerhounds, 30 variants were present in at least one of the chromosome sets, and one variant was present in all 20 sets. The variant present in all chromosome sets is in a gene designated as BMPER (pronounced “bumper”), which is classified as a Tier 2 variant. BMPER stands for Bone Morphogenic Protein-binding Endothelial Regulator. This mouthful of a name is meant to convey that BMPER influences the interaction between a protein that stimulates bone growth (BMP) and the cells that form new blood vessels (endothelial cells). Growth of bone, like the growth of any tissue, cannot occur without the growth of new blood vessels to supply the new tissue.
These initial results in Deerhounds are a mixture of good and bad news. One piece of good news is that three variants are either rare or entirely absent in Deerhounds with osteosarcoma, strongly suggesting that they do not contribute to disease risk in our breed.
Another bit of good news is that, for the great majority of risk variants (30 of 34), a non-risk version also exists in the Deerhound population because it was present in at least one of the 20 chromosome sets from Deerhounds. If a genetic test were developed for any of these 30 risk variants, then the results theoretically could be incorporated into breeding decisions with the aim of reducing the frequency of the risk variant in the Deerhound population. But this theoretical possibility depends upon how common the risk variant is in the Deerhound population.
Ideally, an osteosarcoma risk variant will be common in Deerhounds with osteosarcoma but rare in Deerhounds without osteosarcoma. Breeding to reduce the frequency of such a variant should be relatively easy, in the sense that there would be many potential mates to choose from that lack the variant. On the other hand, if a risk variant is common throughout the breed… well, it becomes harder to reduce the frequency of such a variant. And if the variant is fixed in Deerhounds, meaning it is present in every chromosome set in the population, then we cannot use genetic testing to breed away from the variant.
What about the BMPER gene variant that is present in 100% of chromosome sets from 10 Deerhounds? Even for that one, a non-risk variant may exist in the Deerhound population, based on the fact that a non-risk variant is present in at least some individuals in two Deerhound-related breeds, Greyhounds and Irish Wolfhounds.
A final bit of good news is that, once we understand the mechanism by which a variant increases osteosarcoma risk in Deerhounds – understand which gene is affected and how that change increases cancer risk – then it may turn out the same gene and mechanism are involved in human cancer. This has already proven true for a least a few osteosarcoma-related risk variants in dogs, as Dr. Alvarez’s group pointed out in their 2019 paper. In such situations, drugs developed to treat human cancer by targeting the mechanism also may work in dogs with osteosarcoma. Dr. Alvarez’s group suggested that drugs that inhibit an enzyme involved in some human cancers (designated as CDK4/6) and drugs that block cell-surface receptors involved in some human cancers (designated FGFR1/2) should be tested in dogs with osteosarcoma.
The BMPER gene variant is another one that is important in osteosarcoma in both dogs and human cancer. The BMPER gene’s roles in bone biology and cancer are sufficiently well understood to suggest therapeutic approaches. On top of that, the BMPER gene variant is involved in two processes that make cancer lethal – invasion of adjacent tissues and metastasis to distant sites – making it a high-priority target for anti-cancer drug development. The Alvarez group’s findings suggest that Deerhounds with osteosarcoma offer a potential “animal model” for testing drugs that target this cancer mechanism.
How do we decide which osteosarcoma-related variant(s) to tackle first?
One way to figure out which risk variants are most important in Deerhounds is to rank them by how much they affect risk in general – the tier system that Dr. Alvarez’s group developed. Another way is to rank the risk variants by how often they are present in Deerhounds with osteosarcoma. We don’t know that yet, but we can rank variants by their frequency in the 20 chromosome sets from randomly selected Deerhounds, like this:
|Rank||Frequency in 10 Deerhounds||No. of variants|
|2||80% to 95%||10|
|3||50% to 75%||10|
In an ideal world for Deerhounds, researchers would focus on osteosarcoma risk variants that are both high-tier (have a greater influence on risk) and high-rank (have a greater frequency in Deerhounds with osteosarcoma). But in the real world, researchers will instead focus on risk factors for which the associated gene has been identified and for which variants of that gene have been found to be involved in cancer in humans.
Are there any other osteosarcoma-related genetic variants in Deerhounds?
The short answer is, “probably.” Dr. Jennie Rowell in the Alvarez group performed a preliminary mapping study of Greyhound osteosarcoma with a subset of their cohort that continues to be collected. Using an experimental mapping method, she identified three additional osteosarcoma-related genetic variants in Greyhounds. Two variants do not have other supporting evidence for inherited risk of osteosarcoma in dogs. But the third variant lies within the Scottish Deerhound “OSA1 locus” that was identified as osteosarcoma-related by Phillips and colleagues in 2010. Combining the information from Deerhounds and Greyhounds could reduce the number of candidate genes in the region where the variant lies from dozens to one or two.
Are the three new osteosarcoma-related variants found in Greyhounds present in Deerhounds?
Dr. Alvarez screened the three new osteosarcoma-related variants mapped in Greyhounds in the chromosome sets from the 10 Deerhounds mentioned above. All three variants were present in approximately half of the 20 chromosome sets. That is good news for two reasons. First, it means it is possible to evaluate how much each variant contributes to osteosarcoma risk – that is, to which tier the variant belongs. Second, it means that a non-risk variant exists in the Deerhound population, offering the hope of genetic testing and reducing the frequency of the risk variant over time.
Might genetic variants that affect body size also affect osteosarcoma risk?
A major risk factor for osteosarcoma is body size, and this presumably is a major contributor in Scottish Deerhounds. Most or all of the major genetic variations associated with giant body size in Deerhounds are likely to be fixed in the population.
A new genetic variant associated with long legs was recently identified in a whole genome sequencing study (ESR1 gene long-legs variant; Plassais, Ostrander et al. 2019). This variant is likely to be associated with osteosarcoma risk for the same reason as body size: i.e., both require prolonged bone growth. Dr. Alvarez therefore screened the 20 chromosome sets from Deerhounds for the SNP markers nearest the ESR1 gene to see if a single haplotype (group of markers inherited together) is present and, if so, whether it varies among individual Deerhounds. All 20 chromosome sets had the most highly implicated SNP marker associated with the ESR1 long-legs variant. However, one chromosome of the 20 revealed variation on one side of that marker. The lack of variation over a long region in the other 19 chromosomes suggests either strong selection pressure, recent selection pressure, or a population bottleneck. A candidate functional variation was reported within the ESR1 long-legs variant (in an intron of the gene), but further analysis is necessary to know if any Deerhounds carry the alternative, normal-legs variant of the gene.
It is possible that all Deerhounds carry the long-legs variant of the ESR1 gene, since long legs are one trait that defines the Scottish Deerhound as a breed so would have been selected for over centuries. As we work to reduce the genetic risk of osteosarcoma in Deerhounds, it is important to understand which osteosarcoma risk factors we cannot select against because they have become fixed in the breed (are present in every Deerhound chromosome set) and which osteosarcoma risk factors we cannot select against without changing the nature of the breed in other ways besides cancer risk.
This last point is important. Cancer risk in Deerhounds is influenced by genetic variants that already exist in the breed. Any of those variants might have some useful, desirable, or even essential function during a Deerhound’s life, such as helping it develop while in the womb or grow long legs or a larger heart after birth. Before we select against an osteosarcoma-related variant, it behooves us to understand, if we can, what other function(s) that variant serves in the breed.
Plans for 2021 and beyond
Dr. Alvarez plans to continue these studies in 2021 with a focus on Greyhounds and Scottish Deerhounds. His main goals are to perform additional genetic mapping and validation of genetic markers for prediction of osteosarcoma risk. Among the new developments in the Alvarez Lab, he has begun collaborating with Dr. Heather Huson of Cornell University. Dr. Huson is expert in the development of breeding values for animal selection involving many loci and prioritized traits. He is working with her in this area for another project and believes it will lead to improved application of his group’s genetic findings in Scottish Deerhounds.
ANESTHESIA SENSITIVITIES/ HYPERFIBRINOLYSIS
Research by Dr. Michael Court at Washington State University is studying the genetic causes of several health problems in Deerhounds, including hyperfibrinolysis (delayed post-operative bleeding), hyperthermia (anesthesia and/or stress related), and slow drug metabolism (anesthesia and sedative sensitivity). We have supported Dr. Court both with a direct grant as well as with some of our CHF funds to support this CHF project.
Dr. Court’s work was delayed because of COVID. Here is where things stand:
In 2019, Dr. Court conducted a retrospective case-control genetic association study. DNA samples from 7 cases (dogs who bled after surgery) and 33 controls (dogs who had surgery but didn’t bleed) were obtained either directly from SDCA club members or from samples previously archived in the CHIC DNA repository, North Carolina State University, and the University of Pennsylvania. Seven different candidate genes were sequenced and only one mutation was found that was associated with bleeding. This mutation was present in all 7 (100%) of the cases (3 were homozygous mutant; 4 were heterozygous mutant), while only 7 out of 38 (18%) of the control dogs had the mutation (all 7 dogs were heterozygous mutant). The results indicated that the relative risk for bleeding is 427 times higher in dogs who are homozygous mutant, and 32 times higher in dogs who are heterozygous mutant when compared with dogs who don’t have the mutation.
The 45 dogs were also genotyped for the Factor VII clotting factor mutation found in Deerhounds. The Factor VII mutation was present at equal frequency in both cases and controls, suggesting that this mutation probably does not contribute to delayed postoperative bleeding, although it may be important for intraoperative bleeding.
In humans, which also have this mutation, heterozygotes are often fine for surgeries when they are younger, but some have bleeding problems when they are older. Dr. Court is in the process of seeing whether the same occurs in Deerhounds.
These findings are currently being prepared for peer review and publication. A commercial version of the hyperfibrinolysis gene test will eventually be available.
Editor’s note: This is extremely exciting news for us, but PLEASE remember that this is a treatable and preventable problem: what this test is going to tell us is which dogs are at risk of post-operative bleeding and need treatment with aminocaproic acid or transexamic acid during and/or after surgery or trauma. We are NOT saying that dogs should be removed from the gene pool because they have this mutation. The test is telling us that these dogs will need to be bred to clear dogs so we can reduce the incidence of this mutation in our breed. OUR GENE POOL CANNOT AFFORD TO BE REDUCED ANYMORE. So please: although everyone’s first instinct is to remove dogs with a potentially harmful mutation from breeding programs, we need to change our thinking to just take hyperfibrinolysis status into account when planning breedings—just like Factor VII.
Slow Drug Metabolism
Last year, Dr. Court published a paper identifying what he believes is the main mutation (CYP2B11-H3) that causes slow drug metabolism in Greyhounds. Dr. Court has found that Deerhounds have the same mutation. The link to the paper is here.
That article was based on work done in the lab. Dr. Court is now confirming whether Greyhounds with the mutation metabolize drugs slower than dogs without the mutation.
Dr. Court is still actively recruiting cases for the hyperthermia study. In 2018, using the gene sequencing panel he developed, he discovered that a new mutation in a gene associated with hyperthermia in people was found in several Deerhounds who had experienced hyperthermia after anesthesia. This looks promising, but we need more cases to see whether this mutation is really the cause.
If you have a dog that experienced hyperthermia during surgery or a stressful event, or if you have DNA stored on a dog that experienced hyperthermia and have not already been in touch with Dr. Court, please contact him.
Dr. Court at the Specialty
Dr. Court is the health speaker at the 2021 National Specialty in Richland, Washington, on June 1-5. Please join us for what is going to be a very interesting talk, which will include information on our future new genetic test for hyperfibrinolysis. Dr. Court will also be doing cheek swabs to collect DNA at the show.
Samples Needed: If you have a dog that had an adverse event from a drug used for anesthesia, sedation, or pain relief or experienced delayed post-operative bleeding, please participate in this study. Email Dr. Court for a cheek swab kit.
Four years ago we funded a project for Dr. Paula Henthorn, of the University of Pennsylvania, to continue her work on cystinuria in Deerhounds. She completed that project last year (see the final report in the January/February 2019 Claymore), but her work continues on this condition in our breed.
As reported last year, Dr. Henthorn did not find any protein changes (changes to the actual proteins that form the DNA of the genes) on or next to the two genes that have so far been responsible for every cystinuria mutation found in people (so far, 200+ have been discovered) and dogs. The next step has been to look for DNA variations that may affect the expression of the gene. In other words, we are looking for the parts of the genome that determine where a gene is expressed (what organ and type of cell), when the gene is expressed (anywhere from what time of day to what time of life, or in response to a different gene or environmental condition) and how much of the protein is made. We now know that in addition to changes to the gene’s actual DNA—what most people think of when we talk about mutations—there are things outside the gene itself that affect gene expression. These things are called “epigenetic.” Often, these epigenetic changes are from modifications of specific proteins that surround DNA in the cell. These epigenetic changes can also be sequence dependent. Dr. Henthorn is in the middle of searching the areas around the two genes for changes that might be implicated in Deerhound cystinuria. So far, she has not found anything, but she still has areas to search.
We reported last year that Dr. Henthorn’s colleagues running the PennGen Metabolic Laboratory (Drs. Werner and Casal) have improved the nitroprusside (NP) test that we have been using for years to test our dogs for the presence of cystine in their urine. As we all know, this test has not been 100% predictive, but they are optimistic that the improvements make the test more accurate. However, the proof is in the pudding, as they say, so we will be spending time over the next year gathering information on how accurate the new test is and how it compares to the old test. This may require that owners of dogs that have had NP tests complete yet another survey, but we PROMISE that it will be short and sweet.
So even though our grant has ended, Dr. Henthorn has continued to work on cystinuria in our breed, and we hope that, short of finding the genetic cause, we will at least make more progress in understanding this disease in Deerhounds, and, possibly, dogs in general.
More information about this project can be found at here.
As always, we will be offering free cystinuria testing of urine samples at the 2021 National Specialty. See below for details.
Samples Needed: Dr. Henthorn still needs DNA and urine samples from affected dogs and their relatives. In addition, a few more samples were provided last year for the neutering study, and it still appears as though neutering prevents stone formation.
However, they still need more samples, so if you have a dog that has formed stones or tested positive on a nitroprusside test and are going to neuter the dog, please collect the samples requested so your dog can participate in the neutering study. Directions for sample collection can be found here.
DILATED CARDIOMYOPATHY (DCM)
As you may remember, before COVID struck the SDCA Board approved a project to Holter monitor 100 Deerhounds to learn more about how heart arrhythmias relate to DCM. COVID has stuck a spoke in that wheel logistically, so we are waiting until next year to start that project. We will, of course, keep you posted, and you can always see updates on the DCM Project page on the Deerhound Health web site.
Samples Needed: We have agreed to provide samples of dogs affected with DCM to the U.K. Deerhound Club, as they are continuing our work on the genetics of DCM. We have arranged that CHIC will send them the DNA so all samples can go into our CHIC DNA bank. We are asking that a little more blood than usual (7–10ccs) be sent, so we can send the British researchers all the DNA they need and still have some left for other projects. We can reimburse the $20 CHIC fee for samples submitted for this project—once the sample has been sent, just let Miranda know if you would like this reimbursement. Here are the directions for sending a sample to CHIC.
Testing: Testing to identify heart defects and DCM are recommended health tests for Deerhounds. Breeding stock should have a cardiac ultrasound (echocardiogram) before breeding, and all puppies should have their hearts listened to (auscultation) by a veterinarian before they go to their homes. The club will once again offer cardiac ultrasounds at the 2021 specialty; see below.
To date, four studies have been funded by the Canine Health Foundation’s (CHF) Bloat Initiative, which the SDCA has supported with our CHF Fund monies. All four studies have been completed, and the final reports have been summarized in previous years’ research wrap-up articles. While the CHF was hoping to get a proposal for a new Bloat Initiative project this year, it has been delayed because of COVID. We will continue to stay in touch with the CHF about this initiative. In the meantime, a past Bloat Initiative grant recipient wrote the following article:
Sharp, C. R., Rozanski, E. A., Finn, E., & Borrego, E. J. (2020). The pattern of mortality in dogs with gastric dilatation and volvulus. Journal of Veterinary Emergency and Critical Care. https://doi.org/10.1111/vec.12932
Here is the concluding paragraph:
“Preoperative euthanasia and hence nonsurvival without intent to treat accounted for the majority of GDV mortality in this study. Given the high rate of nonsurvival without intent to treat it is likely that efforts focused at disease prevention will ultimately affect a much greater improvement in overall disease mortality than those focused on improving treatment.”
Also, it means that the majority (83.5%) of dogs actually treated for GDV in this study survived.
Although hairlessness is not a disease and hairless Deerhounds do not have any health problems associated with their hairlessness, we just wanted to remind you that there is a test for the hairless mutation through Mars Wisdom Panel.
In 2014, Lydia Kerridge, a vet student in the United Kingdom, designed a survey for breeders on portosystemic shunts in Deerhounds to learn more about its incidence. Last we heard from Dr. Kerridge and her supervisor, Dr. Penny Watson at Cambridge University, they were in the final stages of preparation for submission to a journal, but we didn’t hear from them this year.
Testing: Bile-acid testing to identify portosystemic shunts is a recommended health test for Deerhounds. All puppies should be tested by their breeders before they leave for their homes, and all breeding stock should be tested as adults if they weren’t tested as puppies. For more information, there are several articles on portosystemic shunts on the Deerhound Health web site.
Samples Needed: If you have a puppy with a shunt, please bank some DNA in the CHIC DNA bank, so when we find an interested researcher we have samples from affected dogs.
FACTOR VII DEFICIENCY MUTATION INFORMATION
The Factor VII mutation is inherited as an autosomal recessive gene, and there is a genetic test for it, so the breeding goal should be to never produce an affected individual. Factor VII clear (no copies of the mutation) dogs can be bred to any dog. Factor VII carriers (one copy of the mutation) and affecteds (two copies of the mutation) should only be bred to Factor VII clear dogs. Remember, as Dr. Paula Henthorn, our cystinuria researcher says, a genetic test should not alter WHO gets bred, only WHO the dog gets BRED TO. It is only through using all of the lines we have that we’ll be able to maximize the effectiveness of the tests and improve the health of our breed, so no line should be eliminated from breeding because of Factor VII genetic status, especially in light of our shrinking gene pool.
Testing: Breeders should know the Factor VII genetic status of their breeding stock and puppies produced so they can inform owners of the Factor VII risk of the puppy they buy. If surgery is done on a Factor-VII-affected dog, please make sure your vet is prepared for potential bleeding problems with plasma from a dog that is normal for Factor VII (note: not every vet carries plasma). Breeders should discuss with their vets the potential risks of breeding a Factor-VII-affected bitch.
The club will once again offer discounted Factor VII testing at the 2021 specialty; see below.
CHIC DNA BANK
Banking DNA samples from our dogs—and keeping the health records updated—continues to be the single most important thing we can do to help our research projects, as most of our researchers have access to our CHIC DNA bank. As you have read above, all of our researchers have used our CHIC DNA Bank samples, as well as shared samples with each other. We couldn’t have made the progress that we have without all of our banked samples and updated health information. We currently have 356 samples in our DNA Bank and several hundred other samples with our researchers.
As always, we will be offering free CHIC DNA bank sample collection at the 2021 specialty; see below for details.
The SDCA’s Deerhound Health web site and affiliated Facebook page usually posts interesting studies, clinical trials, health clinic information, and other health tidbits. We hope to get back to that this year. If you want to receive the blog posts automatically, just click the “Follow” button in the lower right corner of the site. You can also access it from the SDCA web page home page. Here is the Facebook page address.
2021 NATIONAL SPECIALTY SAMPLE COLLECTION
If there is a national specialty in 2021, we will have a health clinic and sample collection there; it’s a great opportunity to catch up on health testing and DNA collection on your dogs at the best price, and we do the shipping. Here’s what we’ll be offering:
CHIC DNA Bank: We will be drawing blood samples for submission to our CHIC DNA Bank. Cost: FREE! The SDCA and OFA underwrite the processing cost.
Cystinuria Study: We will be drawing blood for DNA and accepting urine samples for NP testing to support our cystinuria research. We haven’t confirmed pricing for this year yet, but testing has been FREE (Penn and the SDCA underwrite the processing costs) as long as a blood sample for research is also provided or Penn already has a blood sample from your dog. Even if your dog has had an NP test before, multiple tests are crucial for the research. Urine collection equipment will be available.
Factor VII: Once again, we will be offering discounted Factor VII testing (the SDCA underwrites part of the processing cost).
Cardiac Testing: We will be holding a cardiac clinic on Friday, June 4.
Hyperfibrinolysis: Dr. Michael Court, our health speaker, will offer cheek swab sampling to collect DNA for his research.
For more information, follow the Deerhound health site posts (“Subscribe to Blog via Email” to the right) or go to the SDCA specialty website.
As always, you can bring blood and urine samples to the show from dogs that you are leaving at home.
It has been suggested that we look into sponsoring some research on chronic/recurrent pneumonia in Deerhounds. To do that, we need samples—lots and lots of samples, along with any associated diagnostic tests that may have been run on these affected dogs. For now, we are asking that you put DNA from affected dogs in our CHIC DNA Bank.
The sooner we start actively collecting samples, the sooner we will find a researcher interested in taking this on.
You can always make a donation to the Bunnie Austin Health Fund through the Deerhound Store.
We can’t do all this work without your support. Thanks to everyone who made generous donations of samples and funds last year. And thank you to everyone who shared your stories—you have no idea how much your experiences help our research.
If you have any questions about any of these projects, please don’t hesitate to ask.