LPN questions August 2010

 

1- Age of Onset.

 

The June 17, 2010 announcement states that “Dogs being homozygous (two copies of the mutation) for this mutation (LPN1) will typically develop neuropathy before they reach 3 years of age”.

 

We now have several results that many owners and breeders are finding confusing. For example, a UK bitch was confirmed with laryngeal paralysis, had suffered a collapse of her back legs, and was confirmed for full LPN via nerve biopsy examined by Dr. Shelton, all by the age of 2 ½ years. She has now been confirmed as N/N for LPN1.

 

A male who started experiencing breathing and pelvic leg coordination issues before the end of his first year, had a repair to his cruciate ligament, but went on to what appears to be full blown LPN (not confirmed by biopsy, but very characteristic gait abnormality, laryngeal tie-back procedure, etc)  by 18 months of age. He has also now been confirmed as N/N.

 

The question is: Is it not misleading to suggest that there is any age based distinction between LPN1 and other form(s) when what is currently being called “late onset” LPN can also strike at a very early age?

 

Our intent was not to be misleading. What we do know for sure is that all dogs with 2 copies of the LPN1 mutation develop a severe, early-onset polyneuropathy (as stated, typically by age three years). However, dogs with 2 copies of the LPN1 mutation do not account for all cases of neuropathy, or even all young onset cases. At present, it appears that approximate 60% of the dogs with a biopsy confirmed polyneuropathy and clinical presentation at or before age 4 years have two copies of the LPN1 mutation. An additional 10-15% of biopsy positive dogs with younger-onset clinical signs are heterozygous for the LPN1 mutation. However, 25-30% of these same dogs are actually clear of the LPN1 mutation. Overall, LPN1 mutation homozgyous dogs account for about a quarter of biopsy confirmed neuropathy.

 

The average age of onset in the LPN1 homozygous dogs is about 1.5 years (early-onset). The average age of onset of a polyneuropathy in dogs that do NOT have 2 copies of the LPN1 mutation is 6 years of age. This does not mean that all dogs testing as clear or heterozygous for the LPN1 mutation can only have a late onset polyneuropathy, it means that the AVERAGE age of all dogs is 6 years. The range in age of onset in LPN1 clear and heterozygous dogs is 1.5-11.5.

 

Unfortunately, we don’t know how many different forms of LPN exist. Cases of LPN1 N/N dogs that are affected by early onset LPN symptoms suggest that another early-onset, severe form of LPN exists among Leonbergers.

 

Finally, we selected LPN1 as the name of this first mutation/disease in order to eventually distinguish among types. We have been trying to avoid actually calling this mutation, and any additional mutations that may be discovered, “young-onset” or “late-onset” as an actual disease name, as this can be quite misleading, particularly while we are still matching the data as dogs are genotyped for LPN1 with their phenotype (disease status).

 

 

2- Different mutations, same illness?

 

We have been told that the only way to get a definitive diagnosis for Leonberger Polyneuropathy is through examination of nerve samples at a microscopic level. Here, the very characteristic pattern of this form of neuropathy shows itself clearly and may be distinguished from other forms of neuropathy. We have also been told that the LPN1 mutation is only responsible for one version of this illness, and that other genetic abnormalities likely account for other versions of this illness.

 

The question is: Is it really the case that two spontaneously occurring genetic mutations have coincidentally arisen in our breed, both of which produce nearly identical neurological illnesses, which, on a microscopic level, appear identical?

Similar to Charcot-Marie-Tooth (CMT) disorders in people, it is possible for multiple mutations to present with extremely similar neurological signs and histopathogical changes. As of early 2010, mutations in >35 different genes have been identified as causes of CMT. Histopathological changes in CMT are usually classified as affecting the myelin or the axons of the nerve cells, or, in the case of intermediate forms of CMT, both the myelin and the axons. It is important to obtain a nerve biopsy to confirm that a polyneuropathy truly exists, and that the clinical signs that a dog is presenting with are not being caused by other disease processes, such as spinal disc disease, heart failure, hypothyroidism, etc. Even in families with a history of LPN, other causes of clinical signs need to be ruled out. Finally, while histopathological examination of a nerve biopsy will indeed demonstrate characteristic changes of LPN (or CMT in humans), there are a very limited number of ways in which nervous tissue will respond to insult/injury. Therefore, several neurological diseases may look quite similar or even indistinguishable on a nerve biopsy. This is why it is vital to pair the nerve biopsy with as much additional information about the patient as possible (including clinical signs, any other tests that were performed, etc.).

In the case of the Leonbergers, indeed, it does appear that at least two independent genetic mutations have occurred, and that they do lead to similar neurological illnesses. Although this may sound like a bizarre coincidence, one has to also keep in mind that neurons are probably the most sensitive cells in the body. Therefore many disturbances of general physiological processes can manifest as neurological disease, because the neurons are the first cells that start dying in the body. This reality is confirmed by the dozens of known mutated genes for CMT in human patients. As long as only one causative mutation is known, it is very hard to speculate about common links between the different LPN forms.

 

3- Associated illnesses?

 

We have seen what appears to be an above average pattern of cruciate ligament injuries occurring early on in the lives of Leonbergers, who then go on to develop LPN. We have also seen an apparently above average incidence of digestive issues occurring in LPN dogs. Is it possible that both of these conditions are connected with, or are perhaps exacerbated by, LPN? Does degeneration of the peripheral nerves in the hind legs make it more likely that a dog will damage its cruciate ligament? Does the laryngeal nerve have any association with the vagus nerve that could account for these reported episodes of reflux and dry vomiting?

 

A paper that is currently in press in The Veterinary Journal by Nicolas Granger entitled “Canine inherited motor and sensory neuropathies: An updated classification in 22 breeds and comparison to Charcot-Marie-Tooth disease” states the following: “Orthopaedic lesions can worsen the locomotor deficits and are sometimes considered as primary injuries, whereas they are in fact secondary to the neuropathy (e.g. due to lack of muscle support to the joints).”This seems to suggest exactly what has been suspected: the atrophy of the pelvic musculature may, in fact, predispose the dogs to cruciate rupture and perhaps other orthopedic injuries as well.

 

Gastrointestinal problems have been reported in CMT cases, so it is possible that similar problems could be occurring in Leonbergers with LPN. It is impossible for us to know at this time if the neuropathy extends to the vagal nerve. Historically, the laryngeal nerve(s) were presumed to be involved earlier in the disease process due to the extreme length of the nerve. Similarly, the nerves in the hindlimbs are quite long. It is within the realm of possibility that all nerves in the body are affected to some degree, including the vagus.

 

We cannot, however, comment specifically on these problems as there is not extensive recorded data to match with dogs’ genotypes. The LPN1 gene has no known specific role in either orthopedic or gastrointestinal processes. However, we cannot rule out that there may be links between the other unknown LPN gene(s) and cruciate ligament injuries or digestive issues.

 

4- Future research?

 

Crucially for our breed, how is research on finding the marker for the other version(s) of the disease coming along? It is particularly critical for the future health of Leonberger dogs to understand the genetic causes of later-onset cases, as these dogs will have been bred and passed on their mutations long before anyone suspects that they are affected with LPN.

 

The search for additional LPN mutations is underway and we have strong indications for at least one additional locus on a different chromosome. However, in contrast to the LPN1 gene this second locus is at the stage of identifying potential “LPN2” gene(s) and these genes have not been well characterized in the literature. Therefore, it may still take some time until genetic testing will become available. It is of utmost importance that we continue to receive samples from Leonbergers for our research at both the University of Bern and the University of Minnesota, especially from affected Leonbergers that have a biopsy confirmed diagnosis and are N/N at the LPN1 gene.

 

At the University of Bern and the University of Minnesota, samples submitted from affected Leonbergers that have undergone a neurological examination (which was abnormal) and testing to rule out thyroid disease are candidates for free LPN1 testing. If you have an affected dog that fits these requirements, please email Cord.Droegemueller@itz.unibe.ch in Berne or lpninfo@umn.edu in Minnesota for special shipping instructions.

 

5-Testing Young Puppies?

 

Can puppies younger than 8 weeks be tested by taking cheek swabs? How early can they safely be swabbed and how quickly can the results be made available?

 

At the University of Bern, only blood samples are accepted for genetic testing. We guarantee a turnaround time of three months, but in general the clients get the results faster than that.

At the University of Minnesota: We can test dew claws from the removed tissue of newly whelped pups, and after a pup has been weaned, we can accept cheek swab samples. Ideally, pups should be isolated from the dam and littermates for 1 hour before swabbing. For blood sample testing, the dog should be old enough to have 1-3 ml of blood drawn by a veterinarian. See our webpage at: http://www.vdl.umn.edu/guidelines/canineneuro/home.html for instructions on submitting these samples.

6- LPN1 clears becoming affected with other forms of LPN? 

 

What percentage of returned test identified to the owners as "clear" for LPN1 (N/N) have been given to dogs who actually have been diagnosed or show signs of polyneuropathy and what method is available for these people to query the results?

 

At both universities, the genetic testing is done according to high methodological standards and standard operating procedures (SOPs) are in place. If anyone seriously questions a testing result, the only way of double-checking is the submission of a new blood sample and the repetition of the test.

 

At the University of Minnesota, SOP for the LPN1 test includes running each sample in duplicate and using positive and negative controls in each test batch.

 

At the University of Minnesota, 692 N/N test results have been obtained. Within this group there were 41 (almost 6%) biopsy confirmed polyneuropathy cases and 87 (12.5%) cases with abnormalities that suggested polyneuropathy.

 

At the University of Bern 286 LPN1 N/N test results have been obtained. Within this group there were 8 (3%) confirmed polyneuropathy cases and 17 (6%) suspected polyneuropathy cases.

 

7- Accuracy?

 

Is the test 100% accurate? Have you had any evidence of false positives?

 

This is a 100% specific test for a DNA segment deletion and the test is 100% accurate for detecting this specific DNA segment deletion. There has been no evidence of a false positive to date.

 

So far, all dogs tested D/D at both universities (totalling 30 dogs) have all had clinical signs of polyneuropathy.

 

8- Issues of parentage?

 

What if an affected dog has one or both parents tested clear, does that mean that they are not the parents? What if a dog is tested carrier and both parents tested clear, does that mean at least one of the parents is not the parent?

 

Test results within families indeed provide double-checks on the parentage. If incompatible results are obtained, the reason is either a genotyping error (most likely caused by the mix-up of samples) or a parentage error. According to our experience parentage errors are much more common than genotyping errors.

 

For the examples described in the question above, a clear (N/N) parent CANNOT be the sire/dam of an affected (D/D) dog, because one of the D’s has to come from each parent. Similarly, a carrier dog (D/N) cannot come from two clear (N/N) parents.  

 

9- Previous DNA on file?

 

If DNA is already available in the DNA bank, will the test be cheaper?

 

The University of Bern and the University of Minnesota will provide test results free of charge to all owners who donated samples for research before the 15th of June, 2010. If you have submitted a sample for research before this date and not yet received your free test result, please contact Prof. Cord Drögemüller at Cord.Droegemueller@itz.unibe.ch for the University of Bern or Katie Minor at lpninfo@umn.edu for the University of Minnesota.

 

We will continue to give free LPN1 tests to submitters of blood samples from affected dogs, if they also send us a copy of a qualified abnormal neurological exam together with normal thyroid blood test results, OR the positive result of the histopathological examination of a muscle/nerve biopsy. For the University of Minnesota, if you are interested in submitting your dog’s sample for this free testing, please contact us at lpninfo@umn.edu for special shipping instructions.

 

Should a second “LPN2” test become available, this will be cheaper for dogs which already have DNA banked at the University of Bern. This will likely also be the case at the University of Minnesota.

 

10- Tests for full litters?

 

If for instance cheek swabs (If possible) or blood from several or all pups from one litter are sent at the same time, will the test be cheaper?

 

At the University of Minnesota, we do not offer volume discounts, but we do offer cheek swab and dew claw testing. We can perform the tests as “Rush” samples to ensure a fast turnaround time.

 

At the University of Bern, we cannot accept cheek swabs for testing. We do not give discounts for less than 100 samples.

 

11- Prevalence of the mutation?

 

From info Bern: “According to our studies, currently about 25% of all Leonbergers are carriers of the LPN1 mutation” 

 

Wouldn’t more young Leonbergers be affected if the percentage is that high? Isn’t 25% more likely the average percentage of the dogs participating in the research?

 

If there are 25% LPN1-carriers (D/N), one would expect to see 1.5% - 2% homozygous affected dogs (D/D). This corresponds well with our observations.

 

The number of DD dogs is calculated based on the assumption that any dog has the equal opportunity to breed with any other dog. Therefore, if there is a 25% chance that one dog is a carrier (D/N) and there is a 25% chance that the other dog is also a carrier, then there is only a 6.25% (0.25*0.25) chance that this mating will happen. Then, only ¼ of THOSE offspring will be D/D, which gives the ~1.5% D/D rate. This also assumes that all D/D dogs are showing clinical signs and are not bred to one another.

 

12-Breeding recommendations?

 

The announcement in June included the following comment: “At this time we do not know whether dogs heterozygous for this mutation (one copy of the mutation) might also develop mild clinical signs late in life, but they will most likely not develop severe disease.” In view of the advice not to exclude D/N dogs from breeding, what percentage of these dogs are later developing the disease and showing clinical signs? If it’s only a very low %, then it may be worth the risk of having a later onset disease. But if it’s a very high %, it may not be something to do?

 

We don’t yet have enough data to answer this extremely important question. Until potential other disease-causing mutations are discovered, it is impossible for us to determine why some D/N dogs develop mild disease later in life. It COULD be due to the single copy of the LPN1 deletion, or it could be due to another form of disease (yet to be elucidated), or even a combination of the two.

 

However, eliminating all D/N dogs from breeding will certainly have extremely negative consequences for the genetic diversity of the breed. Therefore, as long as it is not absolutely clear that D/N dogs will indeed all develop neurological disease, we recommend keeping them in the breeding pool for one generation. Litters of D/N x N/N matings should be tested and preferentially the N/N pups (50%) should be kept for future breeding.

 

13- Further samples?

 

We know that the research is continuing in an effort to identify the gene(s) responsible for additional forms of polyneuropathy illness. Do you still need blood samples from additional Leonbergers? Are there any specific lines that are of particular interest for further sampling? Also, do you still seek samples from elderly Leos with no apparent signs of neurological illness?

 

Both universities are still collecting blood samples. As outlined above, we are particularly interested in affected dogs that are clear of the LPN1 deletion (N/N dogs). Therefore, we are offering free testing for affected dogs that submit samples along with copies of all relevant clinical and pathological exams – specifically, an abnormal neurological examination and normal thyroid blood tests with or without biopsy results. The best possible diagnostic test remains a biopsy exam by Dr. Diane Shelton or Prof. Thomas Bilzer. Furthermore, it is of utmost importance that owners who have submitted samples from their dogs in the past keep us updated on any significant changes in the health status of their dog. If a sampled dog dies, please inform us about the exact date and the presumed cause of death.

 

Due to confidentiality, we are not naming specific lines of dogs from which we are interested in obtaining additional samples.

 

At the University of Minnesota, we are not actively seeking samples from elderly Leos clear of neurological signs unless you are also planning to obtain a nerve and muscle biopsy from this dog at post-mortem (to definitively show that the dog’s nerve was normal). These samples will not receive free LPN1 testing. 

 

 

 

14- Pedigree analysis?

 

Now that we have many results coming in, and now that there are more than 800 DNA samples and pedigrees available to the researchers, is anyone doing pedigree analysis of the apparent patterns of inheritance? Is it possible that, through such analysis, one might eventually be able to identify likely lines in the past from which these illnesses may have originated?

 

The pedigree information is taken into consideration during our research. However, given the complexity of the problem (several mutations), it is unlikely that we can provide simple answers.

 

The possibility exists that the LPN1 mutation could eventually be traced back through specific lines, but, again, due to confidentiality, we cannot release this information. We likely never will be able to determine which dog(s) was the founder in whom the mutation(s) originally arose.

 

15- Patterns of inheritance?

 

Although we know that the genetic mutation(s) for other form(s) of LPN are still unknown, there is a very urgent need for breeders to try to avoid affected lines as much as possible. Of particular concern are dogs that develop LPN at an older age, because they were apparently healthy when youngsters, and will have been bred, producing offspring, long before developing outward signs of the disease. Given that your research programs now holds the most complete records of confirmed cases, is it at all possible to give breeders some idea of where the patterns of inheritance are showing up? We understand that naming living dogs, (or even those of the last generation) is not perhaps possible, but can you make any statement at all about what specific historic dogs you are seeing most commonly in the pedigrees of confirmed cases?

 

Given the complexity of polyneuropathy and our limited knowledge of the causative mutations, as well as confidentiality agreements made to each owner submitting samples, this is not possible. We understand the breeders’ deep desire to avoid all LPN in their dogs and we are working on uncovering the genetic causes of these other forms of this disease.

 

16- Spread of the mutation?

 

Do you have any insight into the current rate of spread of the mutation for other forms of LPN through the overall population of Leonbergers (ie, non-LPN1 forms)? In other words, in the absence of any effective test coming into use in the near future, and given the present rate of breeding, how long do we have before the various regional as well as the overall world wide population of Leonbergers is likely to become saturated with the affected gene(s) for non-LPN1 LPN?

 

If there is no selection against other form(s) of LPN, the allele frequency is only influenced by so-called genetic drift within the population (= random event). The frequency can increase, remain at a constant level, or even decrease. The larger the effective population size (avoid inbreeding, avoid “popular sires”, using as many different sires and bitches as possible), the slower these changes will be.