Inherited Leonberger Polyneuropathy.
Dr. Kaspar Matiasek
Neuropathologist,Department of Veterinary Clinical Sciences,
Ludwig-Maximillians University,Munich
This seminar took place in 2007.
Kaspar Matiasek opened his remarks by thanking the LCGB and expressing his appreciation at being able to address a topic that he described as being “somewhere between hysteria and underestimation” in our breed. He compared this disease to some other canine conditions which he feels have been highlighted by some clubs, while at the same time others underemphasized them. He said that in our case, PN in Leonbergers appears to be more widespread than initially thought.
He explained that he would first talk about the theory behind the disease and then go on to demonstrate the outward signs of the condition in a real example, and thanked Sally-Anne Sandalls for bringing in her affected dog for this purpose.
To begin with, Dr. Matiasek set out to give an overview of some of the terminology used in discussing a disorder of this type, and to provide clear definitions of some of these terms.
Predisposition:
From a statistical point of view, a breed that is more prone to the development of a certain disease than others appear to be. Predisposition may be related to the genetic profile of the animal, but also may be due to other factors like construction, such as in the case of orthopaedic problems being more common in large breed dogs. Therefore, predisposition deals only with frequency of occurrence, and, in his words: “Predisposition doesn’t tell you anything about the cause of the disease”.
Familial:
When a condition is more prevalent in related individuals who share some aspect of common ancestry. He adds that “if this term is used it means that the genetic defect has not been determined.” Only the pattern of relationships in affected individuals.
Inherited:
This means that the occurrence of a disease is based on actual DNA alterations within an individual, “which does not necessarily mean that this disease was inherited from the parents, but means that beginning at the point where we find this mutation, that this disease will be inherited in the future”. This is clarified further on in his description of random genetic mutations.
Congenital:
This means simply that this disease has been present in an individual from birth.
Dr. Matiasek then presented a basic introduction to genetic concepts, beginning with the genetic code carried in the DNA of each individual, or Genotype, which is made up mostly by DNA inherited from parents, but also to a lesser extent from mutations to the code within the individual itself.
The term Phenotype is used to describe the appearance of an individual and is not purely derived from the genetic code within, but is also influenced by environmental factors. He uses the example of identical twins who, regardless of having matching DNA or Genotype, develop into distinct individuals physically and behaviourally. “There are several other factors that are influencing the outcome of our cells apart from just the genetic code”. Therefore the Phenotype differs from the Genotype.
There are two ways in which genetic information within the DNA can be modified- from outside and from inside. Outside factors include, for example, harmful radiation such as x-rays that can, over time, have a detrimental impact on an individual’s DNA. The term “Episomal” refers to other, as yet poorly understood internal factors, which can influence an individual’s DNA but which are themselves not the result of pre-existing genetics. All of these factors; the genotype, external influences and so-called Episomal influences, all contribute to the Phenotype – the specific characteristics- of each individual. This is one reason why, even if an individual has a specific genetic defect, it may not develop a disease in exactly the same way, or to the same extent, as another individual with the identical defect. This helps explain why some individuals might be affected early, and some later on in life.
Dr. Matiasek showed us a list of the conditions that our breed has been noted as having a predisposition for:
Hip Dysplasia
Osteochondritis Dissecans
Polydactyllia
Hypothyreosis
Addisons
Eyelid abnormalities
Cataract
Aortic Aneurysm
Laryngeal stenosis / paralysis
Polyneuropathy
These conditions may or may not have specific genetic causes, (none has yet been firmly identified), but there is clear evidence of occurrence within related individuals down through pedigrees, which strongly indicates some degree of underlying genetic cause. Of these conditions, Laryngeal paralysis and Polyneropathy are often found together in affected individuals, but only through microscopic analysis of a nerve tissue sample can they be proven to be related to a single underlying cause. He added that in future he hopes that a genetic test will be available to aid in diagnosis. He clarified by saying “A lot of conditions affect the Laryngeal nerve and thereby cause a collapse of the larynx”. He went on to say that a dog’s laryngeal nerve can be affected by these conditions throughout its life, but that they are more common in elderly dogs because the laryngeal nerve, being the longest in the body, is most susceptible to anything that affects peripheral nerve function, and age is one such factor. However, because LP and PN are very often occurring together, this is one of the reasons why a genetic test is so important “in order o make a clear line between them”. The name of the condition - Polyneuropathy- simply translated, means that multiple nerve fibres are adversely affected. It is generally considered to be bilateral- both sides of the body are equally affected.
History in Leonbergers:
In 2003, Dr. Diane Shelton defined the breed specific version as Inherited Leonberger Polynerupathy. Prior to that, evidence for the disease within the breed goes back at least to the 1950s, when an academic paper was published in Munich describing such a condition in Leonbergers, including descriptions of gait abnormalities. Further examples appear in descriptions of affected dogs in the 60s. Of course there is no clinical proof that these are all cases of the same condition, but the circumstantial evidence is very strong. In the 70s a formal description of the condition was published in the Netherlands, where a university study described Leonbergers with gait abnormalities and laryngeal paralysis being examined under clinical conditions. There follows very little formal evidence other than some vague accounts of similarly affected dogs until a group lead by Diane Shelton undertook the first systematic investigation of the disease in Leonbergers. Samples were collected from many leonbergers and compared. The results of this effort were that for the first time the disorder could be conclusively diagnosed through microscopic evidence of nerve tissue. Since then, there has been a lot of effort put into gathering blood samples for DNA analysis and detailed studies of pedigrees, but little progress has actually been made in terms of furthering our knowledge of this disease.
Diane Shelton has been collaborating with various colleagues including Dr. Patterson, a geneticist based in Minnesota. Similarly, there is a group based in Paris also investigating this condition in leonbergers, (a member of which, Dr. Nicolas Granger, has just moved to Cambridge University here in the UK). Similar efforts continue elsewhere, such as in the Netherlands, and it is hoped that through collaboration, a genetic marker can be located.
The original study by Diane Shelton and colleagues posed the question of whether or not Inherited Polyneuropathy in Leonbergers was connected to a similar condition found in humans called Charcot-Marie-Tooth Disease, (named after the three research scientists who identified that disorder). This condition is estimated to affect one in every 2,500 humans born and, like PN in Leos, is characterised by peripheral muscle and nerve wasting, also accompanied by a loss of tactile sensitivity in the extremities. Despite the relatively high number of individuals within the human population clinically affected with this disease, the way it effects them varies quite widely, with the majority never showing any outside signs of it.
Tracking the disease in Leonbergers:
It is difficult to determine how many cases we have in our breed because there has never been a central registry where all confirmed affected cases are listed.
According to the Leonberger Union, there are estimated to have been about 150 confirmed cases worldwide. It is suggested that somewhere between 2,500 and 4,000 Leonberger puppies are born annually. Based on this, it may well be that the incidence is much less than with Charcot-Marie-Tooth Disease in humans. In fact, the occurrence of Inherited Polyneuropathy in Leonbergers appears to be significantly less than other genetic conditions such as hip dysplasia. A genetic test would allow us to identify affected individuals early enough to eliminate the condition entirely through excluding affected individuals from future breeding programmes. Dr. Matiasek has personally seen confirmed cases from Switzerland, Germany, and probably Austria. Not all confirmed cases have been registered. One reason is that permission is not always granted by the owners of affected dogs. This difficulty can only be resolved by greater transparency and willingness to share information among individual owners and breeders.
Other problems:
Late onset; Some cases in Leonbergers are only confirmed later in life, in some cases after the affected individual has already been used in breeding.
No clarification of the genetic trait, so we have no way to easily determine if the dog carries the affected gene. The only diagnostic test we have is the full nerve biopsy, which is clearly impractical to use on all dogs as a general screening tool.
The outward clinical signs alone are not enough, because there are many other types of polyneuropathies affecting dogs, and many other sorts of conditions that can lead to similar visible symptoms developing.
Therefore, the only confirmation we have at present is the tissue analysis. This involves a general anaesthetic, and a small incision to remove a sample of nerve and muscle, both of which are not without some minor risks.
Effects in Leonbergers:
In the confirmed cases we have, male animals appear more frequently than females, which originally suggested an x-linked mode of inheritance, (which is no longer generally thought to be the case, although recent evidence from Scandinavia again suggests an x-linked mode.) The age of onset can be anywhere from 1- 9 years, with average cases becoming clinically affected in the first 3 years of life. Initial symptoms can be either gait abnormalities, or “dysphonia”, (a change in the dog’s voice and or breathing caused by the affected laryngeal nerve), or both. In early stages, the disease can present as relapsing / remitting, (meaning symptoms come and go), but ultimately the condition is chronic and progressive. The affect on the larynx can lead to pneumonia, which could be another early presenting symptom. If presented with these symptoms, a vet is likely to do a neural examination consisting of two parts: hands-off, which means simply observing the dog’s movement, stance and posture; and hands-on, examining the dog’s mental state, its spinal reflexes, muscle condition, and so forth.
A live demonstration of the outward effects of this condition on a dog was then presented, using “Drummer”, an affected young male Leonberger. In addition to the changes to voice and breathing caused by damage to the laryngeal nerve, Dr. Matiasek described outward signs such a muscle wasting in the lower-thigh area. This is due to a dog’s attempts to maintain a straight knee posture, which relies more on bone position than muscle strength to maintain. This results in a stance in which the affected dog’s hocks are often held in a bent position somewhat forward, under its belly. Dogs so affected will generally “knuckle-over”, meaning they will not immediately correct the placement of their foot if it is folded back so that the pads face up. The gait of an affected dog shows very little flexing of the hock and over-activity in the thigh, resulting in an exaggerated knee action, with the lower leg limply swinging forward, something akin to a puppet being animated by a string attached to it’s thigh. Gait abnormalities in the front legs commonly appear much later than in the rear, but similarly demonstrate weakness and lack of articulation in the lower limb. This disease affects the longest nerves first, the laryngeal nerve being the longest in the body, followed in turn by those that relate to the back feet, those responsible for the front feet, and so forth. Affected dogs may, in addition to these other outward signs, also show some weakness in their facial muscles such as drooping eyelids, due to degeneration of the cranial nerves. In advanced cases, a dog may lose its ability to swallow completely, which, when combined with the inability to close the airways when necessary, can lead to pneumonia.
Diagnosing Inherited Leonberger Polyneuropathy:
The symptoms listed above indicate a neurological disorder effecting the long nerves, but there are many such disorders, (such as metabolic causes for example), so to give a clear diagnosis of Leonberger Polyneuropathy, one must employ more specific diagnostic tests. Electromyography is a minimally invasive procedure that involves testing the electrical conductivity of nerves to muscle in an anaesthetised dog. If the muscle shows no electrical impulse being transferred to it via the nerve, then it is likely that the problem is with the nerve fibres themselves. A full microscopic examination of a nerve tissue sample is then used for final confirmation of the condition. This is a more invasive procedure that involves the removal of samples via a small incision from a fully anaesthetised subject. Even though all other clinical signs indicate Leonberger Polyneuropathy, this final stage is at present the only way to conclusively distinguish it from the many other forms of long-nerve diseases and confirm the diagnosis. Dr. Matiasek showed microscopic examples of affected and normal biopsy samples to demonstrate the degeneration of nerve fibres.
He then showed us a list of similar polyneuropathies known to effect other breeds of dogs, including Malamutes, Dobermanns, German Shepherds, Great Danes etc., along with their suspected genetic modes of inheritance. There is no single gene defect yet known that is common to all. (In contrast, there are many known abnormal genes that cause polyneuropathies in humans and so it is possible to run through a list of known genetic markers when diagnosing similar conditions in humans). The canine genome- the genetic map of DNA- has only recently been fully compiled. Investigations into the specific function of each gene are only now beginning.
In order to identify the affected gene in Leonbergers, it is important in the first instance to study the pedigrees of known affected individuals, in order to discover any apparent patterns of occurrence that might indicate the mode of inheritance. It is not necessary for individual breeders to know the precise gene responsible. What is of vital importance is to understand exactly how it is passed on to future generations. Only then can breeding choices be made that will ultimately eliminate it from the population. (This assumes we have accurate information on the health of ancestors to our breeding dogs). The Leo-Watch group has posted an open registry of confirmed affected dogs on the Internet in order to aid initial pedigree analysis. It is by no means a full list and names only those dogs whose owners have agreed to make them known publicly. It therefore only provides part of the information needed.
Dr. Matiasek then went on to explain some of the challenges involved in identifying the precise abnormal gene, (which is needed to develop a simple genetic marker test). This included explanations of mitochondrial DNA (handed down through the female line only), nuclear DNA, (inherited equally from both parents), Autosomal chromosomes, (equally present in all individuals), sex-linked chromosomes, (which vary between the sexes), acquired mutations, dominant verses recessive modes of inheritance and degrees of penetrance. He also discussed the processes of malfunctioning gene expression, including those that inhibit correct gene function and those that introduce damaging (or toxic) gene expressions.
<This portion of the presentation covered detailed concepts in genetic inheritance, not specific to Inherited Leonberger Polyneuropathy, and so no effort has been made to include full details here. Anyone interested in this technical portion of the seminar may wish to view a DVD recording of the full presentation.>
Dr. Matiasek summarised the current thinking as to the precise mode of transmission of Inherited Leonberger Polyneuropathy. It is believed not to be autosomal dominant. It may be x-linked in some manner, but the most likely mode of inheritance at present is thought to be autosomal recessive. This presents a greater challenge in terms of pedigree analysis because multiple individuals may be healthy carriers of the trait. A further complication arises in the degree to which an individual may or may not be affected by the condition- in technical terms the “penetrance” of the trait – which can vary widely from one individual to another. Also, it may turn out that it is not a single gene that causes the disease, but instead a combination is needed for the condition to arise. Only when the genetic abnormality is correctly isolated, will we be able to employ a simple blood sample to screen an individual’s DNA for the defect.
What we as owners of Leonbergers can do to help achieve this is to send samples for analysis. In the case of a suspected case of ILPN, a thorough clinical examination should be the first step. If the indications of ILPN are strong than a nerve and muscle sample should ideally be collected (can be post mortem) and sent, with the clinical notes, to:
Dr Diane Shelton
Department of Pathology
University of California, San Diego
La Jolla CA
92093-0709
Blood samples for either known or suspected cases should also be sent to:
Dr.Edward Patterson
Department of Small Animal Clinical Sciences
University of Minnesota
St. Paul, Minnesota
The reason that both are necessary is that, at present, only the tissue biopsy can provide a conclusive diagnosis of the condition, but the preservation process (formalin) usually destroys the DNA, so in order to do a DNA analysis of a confirmed case, a separate blood sample is also required. In both cases, the registered name of the dog and its pedigree must accompany the sample. Dr. Matiasek then presented a detailed technical explanation of the procedure for collecting and preserving samples. Again, for brevity’s sake, I have not included this here, but anyone interested may wish to view the complete recording of the seminar.
Where there is a suspected case of full ILPN, a question arose as to whether or not it was better to take a tissue sample from a living dog, or to spare it the invasive procedure by waiting to collect it post-mortem. Dr. Matiasek said that accurate diagnosis was possible with samples from living or deceased individuals. However, an important point to consider is that it might be possible, through analysis of the sample, to determine if the condition is ILPN or, if it might instead be another treatable neurological condition, which is certainly valuable to know during the lifetime of the dog. He stressed that collection of these tissues is a minor procedure, (which does, however require a full anaesthetic), and that in the overwhelming number of cases there are no complications or impairments to the dog’s future quality of life.
Dr. Matiasek then took questions for some time. The following is a brief summary of the main points covered:
Laryngeal paralysis in horses is not necessarily caused by the same disease as Inherited Leonberger Polyneuropathy, but could be due to a number of causes. Likewise, Laryngeal Paralysis in elderly dogs is not simply a sign of old age, but rather, it is a symptom of nerve degeneration which could be due to a number of factors, some age related, other perhaps not. The bottom line is that Laryngeal Paralysis is itself not a disease- it is a symptom, caused by some other factor affecting the long nerve. The impact of LP can be lessened through a procedure called Tie-back surgery, where the paralysed larynx is physically stitched to one side, effectively holding the airway open so as to improve breathing.
Although many conditions may cause a slow degeneration of nerve fibres over time, a microscopic examination of nerve tissue will be able to distinguish ILPN from other conditions. The visible changes are quite distinct, even in cases such as an elderly dog who never showed any outward signs of the condition while alive, but showed the effects of ILPN when a post-mortem sample was examined microscopically. Dr. Matiasek does not however advocate taking post mortem nerve samples from all dogs regardless of whether or not they showed any signs of the condition while alive. Without the necessary clinical tests leading up to the sampling, one could never say for certain what other conditions may have been present. Also, so many samples would quite simply overwhelm any lab attempting to analyse them. Samples from close relatives of known affecteds are however very worthwhile, but again, only if they are accompanied by a complete medical history and full pedigree. However, in the case of simple blood samples gathered from a wide range of leos, they are still very worthwhile –(again if accompanied by a complete pedigree), as they could then be stored for future analysis once some clue as to mode of inheritance is determined. This would essentially become a DNA record of the breed.
There was some discussion about the value of bringing together the many diverse research and diagnostic teams worldwide who are looking into this condition, in the hope that, through closer collaboration, more progress can be made. Similarly, Dr. Matiasek expressed the hope that the German Leonberger Club itself will, in future,
acknowledge the need to be more forthcoming about this disease within their own national population.
The question was then asked about what advice he would offer in terms of making breeding decisions; would he recommend not breeding from any relatives of known affected dogs? Until the mode of inheritance is known, the question cannot be answered. Having said that, direct offspring of known affected dogs would, in his view, be “disastrous” to use, and he suggested that close relatives should likewise be avoided. The condition is old, and has probably been in the breed a long time, so the gene may already be widespread, so avoiding it completely is probably not possible until a specific genetic marker has been isolated. He suggested that the advice of a qualified geneticist should be sought for a more detailed answer.
In terms of how many nerve samples of confirmed cases would be needed to accurately identify the genetic defect, (leading to a simple screening test), he said that the general rule is 500 to 1000 are needed before a reliable genetic screening test could be developed, although it may still be possible to isolate the defective gene with less – if they get lucky. ILPN is a rare disease. In the Neurology department where he is based, “far less than one percent” of the leonbergers they see are affected with this disorder. It is still, however, a serious concern for which a solution needs to be found. So, in conclusion, his advice is “don’t panic” but remember that we are all responsible for the health of the Leonberger breed, and therefore we should take responsibility for sharing in the effort to eradicate it once and for all. Dr. Matiasek concluded his remarks by saying that, despite the comparatively small overall gene pool we have in our breed (and the inter-relationship between the national populations) it is still a relatively healthy breed, which is something to be proud of.
Update-
Report to AKC Canine Health Foundation
August 14, 2009
Grant # 00920
Genetic Basis of Polyneuropathy in Leonbergers
James Mickelson and Ned Patterson
University of Minnesota
Summary of Project Goals: Numerous Leonberger dogs display a distal, symmetrical polyneuropathy with laryngeal paralysis, commonly referred to as Leonberger Polyneuropathy (LPN). The clinical and pathological characteristics of this disorder demonstrate it to be an axonal neuropathy with striking similarities to forms of human Charcot-Marie Tooth (CMT) disease. Although the mode of inheritance of LPN is not yet known, the ratio of biopsy confirmed affected males to biopsy confirmed affected females is approximately 2.5 to 1; the male to female ratio drops to 1.67 to 1 for all dogs showing clinical signs, with or without biopsy confirmation. We have developed an excellent DNA bank of samples from affected and normal dogs in large part due to the efforts of the Leonberger breed clubs and propose to use these samples in a whole genome association analysis with SNP markers to map chromosomal locations of LPN gene loci. We will then identify candidate genes from these chromosomal loci for future sequencing and mutation identification.
Specific Objectives:
1. Continue collection of DNA samples from LPN affected and unaffected Leonberger dogs and continue characterization of their sex and age affection status.
2. Perform a whole genome association analysis of LPN and unaffected Leonbergers with SNP markers.
3. Pursue finer mapping strategies with a higher density of local SNP markers on Leonbergers and related breeds to identify candidate LPN gene loci.
Progress on Objective 1: For the past 2 – 4 years we have isolated DNA from all Leonberger dogs for which blood or paraffin-embedded muscle has been sent. This has resulted in a collection of over 700 DNA samples from dogs with varying levels of phenotyping accuracy and available medical information. This database requires constant phenotyping updates and at this moment 48 dogs (16 females and 32 males) are confirmed as affected by the diagnostic pathology gold standard of nerve abnormalities observed in biopsy analysis performed by Dr Diane Shelton at the University of California San Diego Comparative Neuromuscular Pathology Laboratory. 94 dogs (36 females and 58 males) are symptomatic and “presumed affected”, but have not been confirmed with biopsies. The diagnosis of affected by biopsy includes signs of neurogenic atrophy and loss of nerve fibers in muscle (cranial tibial) and nerve (common peroneal) biopsy specimens. Atrophy can range from singular and small groups of muscle fibers with occasional necrotic fibers undergoing phagocytosis to large groups of atrophic fibers with scattered, pyknotic nuclear clumps. Various stages of demyelination and proliferation are less commonly observed.
We continue to update our database as additional information is reported on the dogs; occasionally this results in a change in their diagnostic status over time that in turn affects the DNA marker analyses. We also formed a collaboration with Drs Tosso Leeb and Cord Drogemuller of the University of Berne in Switzerland. This entails working together in sample collection, and eventually LPN gene mapping and identification from the SNP chip data, and eventual DNA sequencing of positional candidate genes and loci. We have already received DNA from 82 Swiss and German Leonberger dogs in their collection (11 biopsy confirmed by another pathologist, 26 presumed affected and 45 controls); some of these dogs were included on the SNP arrays.
Progress on Objective 2: DNA from a total of 118 Leonberger dogs has been genotyped on Illumina Canine SNP arrays in two cohorts. Data from the first cohort consisted of 26 dogs, all DNA from white blood cells. This was performed in the hopes that a major result could be obtained rapidly. Fourteen of these dogs were affected (9 of them biopsy positive) and 12 controls (six of these biopsy confirmed negative). After analysis of the data, this first sample collection proved to be too small to obtain statistically significant results for the chromosomal location of LPN gene loci. Therefore, we decided to submit an additional collection of samples for genotyping.
The second sample cohort consisted of 96 dogs that were submitted to the genotyping laboratory in early fall of 2008. 92 of these dogs were newly genotyped (4 being repeated from the first cohort). Of this set of 92, 40 samples were from white blood cell DNA (8 were biopsy negative and 20 were biopsy positive), 19 samples were from white blood cell DNA from Switzerland (8 were biopsy positive), and 33 samples were from muscle tissue embedded in paraffin prior to DNA extraction (18 biopsy negative and 14 biopsy positive).
The SNP genotype data on this second cohort of Leonbergers was returned to us in late November 2008 and many interesting statistical tests have been performed. This data was combined with the first cohort of 26 dogs. This larger data set made it possible for loci not detected on the initial sample to become apparent with some statistical significance at the whole genome level. At this time it appears that as many as three different chromosomal locations exist for genes that are significantly associated with LPN. Multiple SNPs spanning 1 – 4 Mb segments on CFA01, CFA12, CFA17 and CFA28 have been identified that have suggestive statistical significance. The relative significance of these loci varies when different populations are included in the analyses. For example, if control females are excluded from the analysis (since females seem to have a later onset and less severe clinical signs, this group is our least definitive phenotypically), then the locus on CFA17 becomes more significant. Nevertheless, we are considering all four of these suggestive loci as worthy of confirmation with additional dogs and SNPs and moved onto Objective 3.
Progress on Objective 3: Work on this objective is in progress. 159 SNP markers have been genotyped on 372 Leonberger DNA samples using the SEQUENOM genotyping platform and the data returned to us in July 2009. These densely-packed SNPs spanned the four loci of most significance identified by the SNP array analysis, including loci on CFA01, CFA12, CFA17, and CFA28. Of the 372 DNA samples, 42 were from muscle tissue embedded in paraffin, 7 were from cheek swabs, and 323 were from white blood cells. 82 of the dogs were from our collaborators in Switzerland, with the remaining 290 from our own sample collection.
The SNPs on the four loci are being used to attempt to narrow down the regions and identify genes that could be subjected to DNA sequencing to find mutations. Initial data analyses confirmed that these regions are all still of high interest; unfortunately, as updates on dogs’ phenotypic status continue to filter in, the exact numbers change slightly with each re-analysis. We hope to begin looking at candidate genes in the very near future. We are also pursuing the line of thought that all of these loci may be contributing, and are considering how they may be interacting. This type of analysis is more complicated than the traditional single mutated gene model.
Presentations: Ekenstedt K, Patterson E, Shelton GD, Minor K, Drögemüller C, Leeb T, Mickelson J. (2009). Distal Symmetrical Polyneuropathy in Leonberger Dogs. LUPA European Human-Canine Comparative Medical Genetics meeting. Uppsala Sweden, May 19, 2009.
Lay Report to Sponsors: We are using the most current gene identification strategy enabled by the canine genome project, termed SNP chips, to find the chromosomal locations of gene(s) causing susceptibility to polyneuropathy in Leonberger dogs (LPN). The ultimate goal is to develop DNA-based tests for susceptibility to LPN that can be used in breeding decisions to help reduce its incidence and potentially for more specific therapies to address the condition.
The good news is that with the outstanding help of the breed clubs in the US and in Europe sufficient samples for the SNP chip gene mapping experiments were obtained. Encouraging owners to have both symptomatic “presumed affected” as well as asymptomatic “presumed normal” dogs biopsied to confirm this status would be a tremendous help to us in these studies; as would annual updates sent to us by owners of dogs in our studies. Other good news is that we now have several good chromosomal locations for where LPN genes might lie. The less good news is that there are several possible chromosomal locations for these LPN genes that are all about equally likely at the present time. This muddies the picture and may make developing a clear LPN susceptibility test more difficult.
An optimistic scenario at the beginning of our studies was that LPN would be a relatively simple trait with only one or two major genes contributing to the condition. At this time we feel it is not likely that LPN is caused by a single gene and we are preparing for the possibility that a genetic explanation for LPN might take several genes and mutations to produce the full disease. Now that the data from Objective 3 is available, this suspicion has held, as all four of the loci continue to look as though they are involved. We will know more when we begin to examine candidate genes in these regions.
Neuropathologist,Department of Veterinary Clinical Sciences,
Ludwig-Maximillians University,Munich
This seminar took place in 2007.
Kaspar Matiasek opened his remarks by thanking the LCGB and expressing his appreciation at being able to address a topic that he described as being “somewhere between hysteria and underestimation” in our breed. He compared this disease to some other canine conditions which he feels have been highlighted by some clubs, while at the same time others underemphasized them. He said that in our case, PN in Leonbergers appears to be more widespread than initially thought.
He explained that he would first talk about the theory behind the disease and then go on to demonstrate the outward signs of the condition in a real example, and thanked Sally-Anne Sandalls for bringing in her affected dog for this purpose.
To begin with, Dr. Matiasek set out to give an overview of some of the terminology used in discussing a disorder of this type, and to provide clear definitions of some of these terms.
Predisposition:
From a statistical point of view, a breed that is more prone to the development of a certain disease than others appear to be. Predisposition may be related to the genetic profile of the animal, but also may be due to other factors like construction, such as in the case of orthopaedic problems being more common in large breed dogs. Therefore, predisposition deals only with frequency of occurrence, and, in his words: “Predisposition doesn’t tell you anything about the cause of the disease”.
Familial:
When a condition is more prevalent in related individuals who share some aspect of common ancestry. He adds that “if this term is used it means that the genetic defect has not been determined.” Only the pattern of relationships in affected individuals.
Inherited:
This means that the occurrence of a disease is based on actual DNA alterations within an individual, “which does not necessarily mean that this disease was inherited from the parents, but means that beginning at the point where we find this mutation, that this disease will be inherited in the future”. This is clarified further on in his description of random genetic mutations.
Congenital:
This means simply that this disease has been present in an individual from birth.
Dr. Matiasek then presented a basic introduction to genetic concepts, beginning with the genetic code carried in the DNA of each individual, or Genotype, which is made up mostly by DNA inherited from parents, but also to a lesser extent from mutations to the code within the individual itself.
The term Phenotype is used to describe the appearance of an individual and is not purely derived from the genetic code within, but is also influenced by environmental factors. He uses the example of identical twins who, regardless of having matching DNA or Genotype, develop into distinct individuals physically and behaviourally. “There are several other factors that are influencing the outcome of our cells apart from just the genetic code”. Therefore the Phenotype differs from the Genotype.
There are two ways in which genetic information within the DNA can be modified- from outside and from inside. Outside factors include, for example, harmful radiation such as x-rays that can, over time, have a detrimental impact on an individual’s DNA. The term “Episomal” refers to other, as yet poorly understood internal factors, which can influence an individual’s DNA but which are themselves not the result of pre-existing genetics. All of these factors; the genotype, external influences and so-called Episomal influences, all contribute to the Phenotype – the specific characteristics- of each individual. This is one reason why, even if an individual has a specific genetic defect, it may not develop a disease in exactly the same way, or to the same extent, as another individual with the identical defect. This helps explain why some individuals might be affected early, and some later on in life.
Dr. Matiasek showed us a list of the conditions that our breed has been noted as having a predisposition for:
Hip Dysplasia
Osteochondritis Dissecans
Polydactyllia
Hypothyreosis
Addisons
Eyelid abnormalities
Cataract
Aortic Aneurysm
Laryngeal stenosis / paralysis
Polyneuropathy
These conditions may or may not have specific genetic causes, (none has yet been firmly identified), but there is clear evidence of occurrence within related individuals down through pedigrees, which strongly indicates some degree of underlying genetic cause. Of these conditions, Laryngeal paralysis and Polyneropathy are often found together in affected individuals, but only through microscopic analysis of a nerve tissue sample can they be proven to be related to a single underlying cause. He added that in future he hopes that a genetic test will be available to aid in diagnosis. He clarified by saying “A lot of conditions affect the Laryngeal nerve and thereby cause a collapse of the larynx”. He went on to say that a dog’s laryngeal nerve can be affected by these conditions throughout its life, but that they are more common in elderly dogs because the laryngeal nerve, being the longest in the body, is most susceptible to anything that affects peripheral nerve function, and age is one such factor. However, because LP and PN are very often occurring together, this is one of the reasons why a genetic test is so important “in order o make a clear line between them”. The name of the condition - Polyneuropathy- simply translated, means that multiple nerve fibres are adversely affected. It is generally considered to be bilateral- both sides of the body are equally affected.
History in Leonbergers:
In 2003, Dr. Diane Shelton defined the breed specific version as Inherited Leonberger Polynerupathy. Prior to that, evidence for the disease within the breed goes back at least to the 1950s, when an academic paper was published in Munich describing such a condition in Leonbergers, including descriptions of gait abnormalities. Further examples appear in descriptions of affected dogs in the 60s. Of course there is no clinical proof that these are all cases of the same condition, but the circumstantial evidence is very strong. In the 70s a formal description of the condition was published in the Netherlands, where a university study described Leonbergers with gait abnormalities and laryngeal paralysis being examined under clinical conditions. There follows very little formal evidence other than some vague accounts of similarly affected dogs until a group lead by Diane Shelton undertook the first systematic investigation of the disease in Leonbergers. Samples were collected from many leonbergers and compared. The results of this effort were that for the first time the disorder could be conclusively diagnosed through microscopic evidence of nerve tissue. Since then, there has been a lot of effort put into gathering blood samples for DNA analysis and detailed studies of pedigrees, but little progress has actually been made in terms of furthering our knowledge of this disease.
Diane Shelton has been collaborating with various colleagues including Dr. Patterson, a geneticist based in Minnesota. Similarly, there is a group based in Paris also investigating this condition in leonbergers, (a member of which, Dr. Nicolas Granger, has just moved to Cambridge University here in the UK). Similar efforts continue elsewhere, such as in the Netherlands, and it is hoped that through collaboration, a genetic marker can be located.
The original study by Diane Shelton and colleagues posed the question of whether or not Inherited Polyneuropathy in Leonbergers was connected to a similar condition found in humans called Charcot-Marie-Tooth Disease, (named after the three research scientists who identified that disorder). This condition is estimated to affect one in every 2,500 humans born and, like PN in Leos, is characterised by peripheral muscle and nerve wasting, also accompanied by a loss of tactile sensitivity in the extremities. Despite the relatively high number of individuals within the human population clinically affected with this disease, the way it effects them varies quite widely, with the majority never showing any outside signs of it.
Tracking the disease in Leonbergers:
It is difficult to determine how many cases we have in our breed because there has never been a central registry where all confirmed affected cases are listed.
According to the Leonberger Union, there are estimated to have been about 150 confirmed cases worldwide. It is suggested that somewhere between 2,500 and 4,000 Leonberger puppies are born annually. Based on this, it may well be that the incidence is much less than with Charcot-Marie-Tooth Disease in humans. In fact, the occurrence of Inherited Polyneuropathy in Leonbergers appears to be significantly less than other genetic conditions such as hip dysplasia. A genetic test would allow us to identify affected individuals early enough to eliminate the condition entirely through excluding affected individuals from future breeding programmes. Dr. Matiasek has personally seen confirmed cases from Switzerland, Germany, and probably Austria. Not all confirmed cases have been registered. One reason is that permission is not always granted by the owners of affected dogs. This difficulty can only be resolved by greater transparency and willingness to share information among individual owners and breeders.
Other problems:
Late onset; Some cases in Leonbergers are only confirmed later in life, in some cases after the affected individual has already been used in breeding.
No clarification of the genetic trait, so we have no way to easily determine if the dog carries the affected gene. The only diagnostic test we have is the full nerve biopsy, which is clearly impractical to use on all dogs as a general screening tool.
The outward clinical signs alone are not enough, because there are many other types of polyneuropathies affecting dogs, and many other sorts of conditions that can lead to similar visible symptoms developing.
Therefore, the only confirmation we have at present is the tissue analysis. This involves a general anaesthetic, and a small incision to remove a sample of nerve and muscle, both of which are not without some minor risks.
Effects in Leonbergers:
In the confirmed cases we have, male animals appear more frequently than females, which originally suggested an x-linked mode of inheritance, (which is no longer generally thought to be the case, although recent evidence from Scandinavia again suggests an x-linked mode.) The age of onset can be anywhere from 1- 9 years, with average cases becoming clinically affected in the first 3 years of life. Initial symptoms can be either gait abnormalities, or “dysphonia”, (a change in the dog’s voice and or breathing caused by the affected laryngeal nerve), or both. In early stages, the disease can present as relapsing / remitting, (meaning symptoms come and go), but ultimately the condition is chronic and progressive. The affect on the larynx can lead to pneumonia, which could be another early presenting symptom. If presented with these symptoms, a vet is likely to do a neural examination consisting of two parts: hands-off, which means simply observing the dog’s movement, stance and posture; and hands-on, examining the dog’s mental state, its spinal reflexes, muscle condition, and so forth.
A live demonstration of the outward effects of this condition on a dog was then presented, using “Drummer”, an affected young male Leonberger. In addition to the changes to voice and breathing caused by damage to the laryngeal nerve, Dr. Matiasek described outward signs such a muscle wasting in the lower-thigh area. This is due to a dog’s attempts to maintain a straight knee posture, which relies more on bone position than muscle strength to maintain. This results in a stance in which the affected dog’s hocks are often held in a bent position somewhat forward, under its belly. Dogs so affected will generally “knuckle-over”, meaning they will not immediately correct the placement of their foot if it is folded back so that the pads face up. The gait of an affected dog shows very little flexing of the hock and over-activity in the thigh, resulting in an exaggerated knee action, with the lower leg limply swinging forward, something akin to a puppet being animated by a string attached to it’s thigh. Gait abnormalities in the front legs commonly appear much later than in the rear, but similarly demonstrate weakness and lack of articulation in the lower limb. This disease affects the longest nerves first, the laryngeal nerve being the longest in the body, followed in turn by those that relate to the back feet, those responsible for the front feet, and so forth. Affected dogs may, in addition to these other outward signs, also show some weakness in their facial muscles such as drooping eyelids, due to degeneration of the cranial nerves. In advanced cases, a dog may lose its ability to swallow completely, which, when combined with the inability to close the airways when necessary, can lead to pneumonia.
Diagnosing Inherited Leonberger Polyneuropathy:
The symptoms listed above indicate a neurological disorder effecting the long nerves, but there are many such disorders, (such as metabolic causes for example), so to give a clear diagnosis of Leonberger Polyneuropathy, one must employ more specific diagnostic tests. Electromyography is a minimally invasive procedure that involves testing the electrical conductivity of nerves to muscle in an anaesthetised dog. If the muscle shows no electrical impulse being transferred to it via the nerve, then it is likely that the problem is with the nerve fibres themselves. A full microscopic examination of a nerve tissue sample is then used for final confirmation of the condition. This is a more invasive procedure that involves the removal of samples via a small incision from a fully anaesthetised subject. Even though all other clinical signs indicate Leonberger Polyneuropathy, this final stage is at present the only way to conclusively distinguish it from the many other forms of long-nerve diseases and confirm the diagnosis. Dr. Matiasek showed microscopic examples of affected and normal biopsy samples to demonstrate the degeneration of nerve fibres.
He then showed us a list of similar polyneuropathies known to effect other breeds of dogs, including Malamutes, Dobermanns, German Shepherds, Great Danes etc., along with their suspected genetic modes of inheritance. There is no single gene defect yet known that is common to all. (In contrast, there are many known abnormal genes that cause polyneuropathies in humans and so it is possible to run through a list of known genetic markers when diagnosing similar conditions in humans). The canine genome- the genetic map of DNA- has only recently been fully compiled. Investigations into the specific function of each gene are only now beginning.
In order to identify the affected gene in Leonbergers, it is important in the first instance to study the pedigrees of known affected individuals, in order to discover any apparent patterns of occurrence that might indicate the mode of inheritance. It is not necessary for individual breeders to know the precise gene responsible. What is of vital importance is to understand exactly how it is passed on to future generations. Only then can breeding choices be made that will ultimately eliminate it from the population. (This assumes we have accurate information on the health of ancestors to our breeding dogs). The Leo-Watch group has posted an open registry of confirmed affected dogs on the Internet in order to aid initial pedigree analysis. It is by no means a full list and names only those dogs whose owners have agreed to make them known publicly. It therefore only provides part of the information needed.
Dr. Matiasek then went on to explain some of the challenges involved in identifying the precise abnormal gene, (which is needed to develop a simple genetic marker test). This included explanations of mitochondrial DNA (handed down through the female line only), nuclear DNA, (inherited equally from both parents), Autosomal chromosomes, (equally present in all individuals), sex-linked chromosomes, (which vary between the sexes), acquired mutations, dominant verses recessive modes of inheritance and degrees of penetrance. He also discussed the processes of malfunctioning gene expression, including those that inhibit correct gene function and those that introduce damaging (or toxic) gene expressions.
<This portion of the presentation covered detailed concepts in genetic inheritance, not specific to Inherited Leonberger Polyneuropathy, and so no effort has been made to include full details here. Anyone interested in this technical portion of the seminar may wish to view a DVD recording of the full presentation.>
Dr. Matiasek summarised the current thinking as to the precise mode of transmission of Inherited Leonberger Polyneuropathy. It is believed not to be autosomal dominant. It may be x-linked in some manner, but the most likely mode of inheritance at present is thought to be autosomal recessive. This presents a greater challenge in terms of pedigree analysis because multiple individuals may be healthy carriers of the trait. A further complication arises in the degree to which an individual may or may not be affected by the condition- in technical terms the “penetrance” of the trait – which can vary widely from one individual to another. Also, it may turn out that it is not a single gene that causes the disease, but instead a combination is needed for the condition to arise. Only when the genetic abnormality is correctly isolated, will we be able to employ a simple blood sample to screen an individual’s DNA for the defect.
What we as owners of Leonbergers can do to help achieve this is to send samples for analysis. In the case of a suspected case of ILPN, a thorough clinical examination should be the first step. If the indications of ILPN are strong than a nerve and muscle sample should ideally be collected (can be post mortem) and sent, with the clinical notes, to:
Dr Diane Shelton
Department of Pathology
University of California, San Diego
La Jolla CA
92093-0709
Blood samples for either known or suspected cases should also be sent to:
Dr.Edward Patterson
Department of Small Animal Clinical Sciences
University of Minnesota
St. Paul, Minnesota
The reason that both are necessary is that, at present, only the tissue biopsy can provide a conclusive diagnosis of the condition, but the preservation process (formalin) usually destroys the DNA, so in order to do a DNA analysis of a confirmed case, a separate blood sample is also required. In both cases, the registered name of the dog and its pedigree must accompany the sample. Dr. Matiasek then presented a detailed technical explanation of the procedure for collecting and preserving samples. Again, for brevity’s sake, I have not included this here, but anyone interested may wish to view the complete recording of the seminar.
Where there is a suspected case of full ILPN, a question arose as to whether or not it was better to take a tissue sample from a living dog, or to spare it the invasive procedure by waiting to collect it post-mortem. Dr. Matiasek said that accurate diagnosis was possible with samples from living or deceased individuals. However, an important point to consider is that it might be possible, through analysis of the sample, to determine if the condition is ILPN or, if it might instead be another treatable neurological condition, which is certainly valuable to know during the lifetime of the dog. He stressed that collection of these tissues is a minor procedure, (which does, however require a full anaesthetic), and that in the overwhelming number of cases there are no complications or impairments to the dog’s future quality of life.
Dr. Matiasek then took questions for some time. The following is a brief summary of the main points covered:
Laryngeal paralysis in horses is not necessarily caused by the same disease as Inherited Leonberger Polyneuropathy, but could be due to a number of causes. Likewise, Laryngeal Paralysis in elderly dogs is not simply a sign of old age, but rather, it is a symptom of nerve degeneration which could be due to a number of factors, some age related, other perhaps not. The bottom line is that Laryngeal Paralysis is itself not a disease- it is a symptom, caused by some other factor affecting the long nerve. The impact of LP can be lessened through a procedure called Tie-back surgery, where the paralysed larynx is physically stitched to one side, effectively holding the airway open so as to improve breathing.
Although many conditions may cause a slow degeneration of nerve fibres over time, a microscopic examination of nerve tissue will be able to distinguish ILPN from other conditions. The visible changes are quite distinct, even in cases such as an elderly dog who never showed any outward signs of the condition while alive, but showed the effects of ILPN when a post-mortem sample was examined microscopically. Dr. Matiasek does not however advocate taking post mortem nerve samples from all dogs regardless of whether or not they showed any signs of the condition while alive. Without the necessary clinical tests leading up to the sampling, one could never say for certain what other conditions may have been present. Also, so many samples would quite simply overwhelm any lab attempting to analyse them. Samples from close relatives of known affecteds are however very worthwhile, but again, only if they are accompanied by a complete medical history and full pedigree. However, in the case of simple blood samples gathered from a wide range of leos, they are still very worthwhile –(again if accompanied by a complete pedigree), as they could then be stored for future analysis once some clue as to mode of inheritance is determined. This would essentially become a DNA record of the breed.
There was some discussion about the value of bringing together the many diverse research and diagnostic teams worldwide who are looking into this condition, in the hope that, through closer collaboration, more progress can be made. Similarly, Dr. Matiasek expressed the hope that the German Leonberger Club itself will, in future,
acknowledge the need to be more forthcoming about this disease within their own national population.
The question was then asked about what advice he would offer in terms of making breeding decisions; would he recommend not breeding from any relatives of known affected dogs? Until the mode of inheritance is known, the question cannot be answered. Having said that, direct offspring of known affected dogs would, in his view, be “disastrous” to use, and he suggested that close relatives should likewise be avoided. The condition is old, and has probably been in the breed a long time, so the gene may already be widespread, so avoiding it completely is probably not possible until a specific genetic marker has been isolated. He suggested that the advice of a qualified geneticist should be sought for a more detailed answer.
In terms of how many nerve samples of confirmed cases would be needed to accurately identify the genetic defect, (leading to a simple screening test), he said that the general rule is 500 to 1000 are needed before a reliable genetic screening test could be developed, although it may still be possible to isolate the defective gene with less – if they get lucky. ILPN is a rare disease. In the Neurology department where he is based, “far less than one percent” of the leonbergers they see are affected with this disorder. It is still, however, a serious concern for which a solution needs to be found. So, in conclusion, his advice is “don’t panic” but remember that we are all responsible for the health of the Leonberger breed, and therefore we should take responsibility for sharing in the effort to eradicate it once and for all. Dr. Matiasek concluded his remarks by saying that, despite the comparatively small overall gene pool we have in our breed (and the inter-relationship between the national populations) it is still a relatively healthy breed, which is something to be proud of.
Update-
Report to AKC Canine Health Foundation
August 14, 2009
Grant # 00920
Genetic Basis of Polyneuropathy in Leonbergers
James Mickelson and Ned Patterson
University of Minnesota
Summary of Project Goals: Numerous Leonberger dogs display a distal, symmetrical polyneuropathy with laryngeal paralysis, commonly referred to as Leonberger Polyneuropathy (LPN). The clinical and pathological characteristics of this disorder demonstrate it to be an axonal neuropathy with striking similarities to forms of human Charcot-Marie Tooth (CMT) disease. Although the mode of inheritance of LPN is not yet known, the ratio of biopsy confirmed affected males to biopsy confirmed affected females is approximately 2.5 to 1; the male to female ratio drops to 1.67 to 1 for all dogs showing clinical signs, with or without biopsy confirmation. We have developed an excellent DNA bank of samples from affected and normal dogs in large part due to the efforts of the Leonberger breed clubs and propose to use these samples in a whole genome association analysis with SNP markers to map chromosomal locations of LPN gene loci. We will then identify candidate genes from these chromosomal loci for future sequencing and mutation identification.
Specific Objectives:
1. Continue collection of DNA samples from LPN affected and unaffected Leonberger dogs and continue characterization of their sex and age affection status.
2. Perform a whole genome association analysis of LPN and unaffected Leonbergers with SNP markers.
3. Pursue finer mapping strategies with a higher density of local SNP markers on Leonbergers and related breeds to identify candidate LPN gene loci.
Progress on Objective 1: For the past 2 – 4 years we have isolated DNA from all Leonberger dogs for which blood or paraffin-embedded muscle has been sent. This has resulted in a collection of over 700 DNA samples from dogs with varying levels of phenotyping accuracy and available medical information. This database requires constant phenotyping updates and at this moment 48 dogs (16 females and 32 males) are confirmed as affected by the diagnostic pathology gold standard of nerve abnormalities observed in biopsy analysis performed by Dr Diane Shelton at the University of California San Diego Comparative Neuromuscular Pathology Laboratory. 94 dogs (36 females and 58 males) are symptomatic and “presumed affected”, but have not been confirmed with biopsies. The diagnosis of affected by biopsy includes signs of neurogenic atrophy and loss of nerve fibers in muscle (cranial tibial) and nerve (common peroneal) biopsy specimens. Atrophy can range from singular and small groups of muscle fibers with occasional necrotic fibers undergoing phagocytosis to large groups of atrophic fibers with scattered, pyknotic nuclear clumps. Various stages of demyelination and proliferation are less commonly observed.
We continue to update our database as additional information is reported on the dogs; occasionally this results in a change in their diagnostic status over time that in turn affects the DNA marker analyses. We also formed a collaboration with Drs Tosso Leeb and Cord Drogemuller of the University of Berne in Switzerland. This entails working together in sample collection, and eventually LPN gene mapping and identification from the SNP chip data, and eventual DNA sequencing of positional candidate genes and loci. We have already received DNA from 82 Swiss and German Leonberger dogs in their collection (11 biopsy confirmed by another pathologist, 26 presumed affected and 45 controls); some of these dogs were included on the SNP arrays.
Progress on Objective 2: DNA from a total of 118 Leonberger dogs has been genotyped on Illumina Canine SNP arrays in two cohorts. Data from the first cohort consisted of 26 dogs, all DNA from white blood cells. This was performed in the hopes that a major result could be obtained rapidly. Fourteen of these dogs were affected (9 of them biopsy positive) and 12 controls (six of these biopsy confirmed negative). After analysis of the data, this first sample collection proved to be too small to obtain statistically significant results for the chromosomal location of LPN gene loci. Therefore, we decided to submit an additional collection of samples for genotyping.
The second sample cohort consisted of 96 dogs that were submitted to the genotyping laboratory in early fall of 2008. 92 of these dogs were newly genotyped (4 being repeated from the first cohort). Of this set of 92, 40 samples were from white blood cell DNA (8 were biopsy negative and 20 were biopsy positive), 19 samples were from white blood cell DNA from Switzerland (8 were biopsy positive), and 33 samples were from muscle tissue embedded in paraffin prior to DNA extraction (18 biopsy negative and 14 biopsy positive).
The SNP genotype data on this second cohort of Leonbergers was returned to us in late November 2008 and many interesting statistical tests have been performed. This data was combined with the first cohort of 26 dogs. This larger data set made it possible for loci not detected on the initial sample to become apparent with some statistical significance at the whole genome level. At this time it appears that as many as three different chromosomal locations exist for genes that are significantly associated with LPN. Multiple SNPs spanning 1 – 4 Mb segments on CFA01, CFA12, CFA17 and CFA28 have been identified that have suggestive statistical significance. The relative significance of these loci varies when different populations are included in the analyses. For example, if control females are excluded from the analysis (since females seem to have a later onset and less severe clinical signs, this group is our least definitive phenotypically), then the locus on CFA17 becomes more significant. Nevertheless, we are considering all four of these suggestive loci as worthy of confirmation with additional dogs and SNPs and moved onto Objective 3.
Progress on Objective 3: Work on this objective is in progress. 159 SNP markers have been genotyped on 372 Leonberger DNA samples using the SEQUENOM genotyping platform and the data returned to us in July 2009. These densely-packed SNPs spanned the four loci of most significance identified by the SNP array analysis, including loci on CFA01, CFA12, CFA17, and CFA28. Of the 372 DNA samples, 42 were from muscle tissue embedded in paraffin, 7 were from cheek swabs, and 323 were from white blood cells. 82 of the dogs were from our collaborators in Switzerland, with the remaining 290 from our own sample collection.
The SNPs on the four loci are being used to attempt to narrow down the regions and identify genes that could be subjected to DNA sequencing to find mutations. Initial data analyses confirmed that these regions are all still of high interest; unfortunately, as updates on dogs’ phenotypic status continue to filter in, the exact numbers change slightly with each re-analysis. We hope to begin looking at candidate genes in the very near future. We are also pursuing the line of thought that all of these loci may be contributing, and are considering how they may be interacting. This type of analysis is more complicated than the traditional single mutated gene model.
Presentations: Ekenstedt K, Patterson E, Shelton GD, Minor K, Drögemüller C, Leeb T, Mickelson J. (2009). Distal Symmetrical Polyneuropathy in Leonberger Dogs. LUPA European Human-Canine Comparative Medical Genetics meeting. Uppsala Sweden, May 19, 2009.
Lay Report to Sponsors: We are using the most current gene identification strategy enabled by the canine genome project, termed SNP chips, to find the chromosomal locations of gene(s) causing susceptibility to polyneuropathy in Leonberger dogs (LPN). The ultimate goal is to develop DNA-based tests for susceptibility to LPN that can be used in breeding decisions to help reduce its incidence and potentially for more specific therapies to address the condition.
The good news is that with the outstanding help of the breed clubs in the US and in Europe sufficient samples for the SNP chip gene mapping experiments were obtained. Encouraging owners to have both symptomatic “presumed affected” as well as asymptomatic “presumed normal” dogs biopsied to confirm this status would be a tremendous help to us in these studies; as would annual updates sent to us by owners of dogs in our studies. Other good news is that we now have several good chromosomal locations for where LPN genes might lie. The less good news is that there are several possible chromosomal locations for these LPN genes that are all about equally likely at the present time. This muddies the picture and may make developing a clear LPN susceptibility test more difficult.
An optimistic scenario at the beginning of our studies was that LPN would be a relatively simple trait with only one or two major genes contributing to the condition. At this time we feel it is not likely that LPN is caused by a single gene and we are preparing for the possibility that a genetic explanation for LPN might take several genes and mutations to produce the full disease. Now that the data from Objective 3 is available, this suspicion has held, as all four of the loci continue to look as though they are involved. We will know more when we begin to examine candidate genes in these regions.