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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 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 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 At the 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 At the At the 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 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 Should a second “LPN2” test become available, this will be cheaper for dogs
which already have DNA banked at the 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 At the 11- Prevalence of the mutation? From info 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.
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