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About the Health Education Committee - ABTC |2013

Developing a Genetic Marker

History of the Study

The following article was written by Anita M. Oberbauer, Ph.D. and originally published in the American Belgian Tervuren Club's newsletter, the TN

In the June/July 1996 TNT, Dr. Thomas Famula presented a summary of results from a 1986 seizure survey sent to all ABTC members. The impetus behind the ABTC survey at that time was to provide breeders of Belgian Tervuren with information about the heredity of epileptic seizures. The data languished for a number of years, before being sent to Dr. Famula, a quantitative geneticist, for evaluation.

The data of 997 unique Tervuren were first analyzed with the objective to quantify the role of inheritance in onset of seizuring activity. As previously reported in the TNT, the incidence of Tervuren seizures, as determined from the survey data (which included three generation pedigrees), was 17% with a high heritability estimate (Famula et al., 1997).

Heritability

Heritability estimates provide a basis to determine if genetic selection programs (i.e., concerted selected breeding) can impact the prevalence of a particular trait. Heritability estimates reflect a composite of environmental influences (such as diet, temperature, exercise) and genetic influences. In other words, heritability estimates have predictive value as to the likelihood that improvement or change can be achieved by using specific breeding stock.

To put the heritability estimate of Tervuren seizure activity in perspective, reproductive traits, such as how many puppies will be born for a given dam, are difficult to select for and achieve measurable results from one generation to the next. Those traits, which are highly influenced by the environment, have low heritability values in the range of 0.15 or lower.

In reproductive traits, the environmental influences swamp out any detectable genetic influences. In contrast, growth traits, such as height, have relatively high heritabilities (~0.4) and significant progress (increased height) can be made by breeding animals of the desired height. The genetic influence on expression of a growth trait is relatively great and therefore one can exert more effective selection pressure.

Does any trait have a heritability of 1.0? Or rather, what trait is fully governed by the underlying genes and not influenced by the environment? In mammals, the sex of an animal has a heritability of 1.0; a normal male is a male if he carries the Y chromosome regardless of how well the mother ate or exercised during her pregnancy.

The heritability estimates for traits therefore give an indication of how much of the trait of interest is governed by genetics. Heritability estimates are frequently given as a range due to the imprecision of the data, calculations, underlying assumptions, or environmental influences on a particular population under analysis.

The range of the heritability estimate for seizuring in Belgian Tervuren is 0.65-0.88. In other words, highly heritable.

Estimates in that range indicate that significant progress can be made to reduce the incidence of seizures in Tervuren merely by judicious breeding.

What Can Breeders Do?

However, in the absence of an extensive database describing and characterizing breeding animals on their likelihood of carrying or passing on the seizuring condition, what can concerned breeders do?

The data from the Belgian Tervuren survey was additionally analyzed to directly address this concern and is published (Famula and Oberbauer, 1998). To paraphrase this study, by using statistical sampling methods, the probability of producing seizuring offspring can only be estimated when the onlycriteria available for use in selecting breeding animals is the seizuring status of those parents.

Clearly, this is not the ideal way to go about developing selective breeding programs in an attempt to reduce the incidence of this debilitating disorder.

The Single Gene Theory

Analysis of the Belgian Tervuren data (Famula et al., 1997), although clearly indicating a highly heritable basis to seizures, has ruled out a single locus model of inheritance and a supported a polygenic (multilocus) model.

However, heritability estimates of the magnitude detected for Tervuren indicates a single locus/gene that significantly influences the expression of the trait. That is the situation with the Tervuren and seizures.

The data was analyzed by six different methods to assess the presence of a major gene that plays an important, though not exclusive, role in the incidence of epilepsy. While no single one of these tests can be considered conclusive proof that there is a single gene of major effect on seizures, the fact that all six independent tests point to the existence of a major gene, strongly indicates that one major gene does exist.

Seizuring activity due to this major gene appears to be an autosomal recessive.

We have provided an analogy to illustrate how a single gene, though not fully determinant in development of the trait, can significantly influence the outcome.

Developing a Marker

Loci of large effect (major genes) have been utilized in other species for the development of genetic markers to assist breeders in selecting breeding stock that are less likely to pass on the genetic disorder. We will utilize the rapidly developing canine genome map and polymorphic canine markers to develop a linkage between a marker and the seizure phenotype for the Belgian Tervuren.

The most difficult aspect of developing a marker linked to some genetic disorder is identifying the mapping population. A mapping population refers to the individuals that are screened for an association between a genetic marker and the disorder.

Mapping Population

The mapping population is the most critical consideration. Ideally, we wish to minimize the background noise due to natural variation among individual dogs that is totally unrelated to seizuring. Every animal is different from every other animal. While this difference is to be expected, we do not want to get distracted by differences that have nothingto do with seizures.

For instance, if we evaluated every Belgian Tervuren that its owner classified as being epileptic, it would take years to wade through all the natural variations that have just accumulated by chance (ear set high, ear set low, coat semi-coarse, coat soft, coat a bit shorter than other Tervs, and so forth) in our search for the specific variation linked to seizures. We wish to do a directed search on a sub-population of highly related dogs as they will have less natural variation than the entire population of Tervuren.

Once that marker linkage to seizuring is established in the subpopulation, its general applicability to the entire Tervuren population will be tested.

So what is needed?

We need those subpopulations (families).

And that is where the ABTC membership can help! We need DNA from families of Belgian Tervuren known to be affected by seizures, not just dogs that are classified as epileptic.

What is the definition of a family?

  •  dog or bitch known to pass on seizuring activity
  • offspring from these dogs or bitches
  • dogs and bitches these dogs were bred to (even if the breeding did not produce any seizuring offspring)
  • parents of either the dog or bitch
  • siblings of either the dog or bitch
  • repeat breedings of dogs or bitches that have produced seizures, even if no seizures were produced by the repeat breeding dogs or bitches

We need to know the seizuring status of dogs submitted and also, the parents and offspring (as well as can be defined). The seizuring documentation is critical to assign phenotype. Veterinary records substantiating seizures are great.

This study is not limited to dogs within the United States. We encourage breeders from Canada and Europe to contribute to the studies with families afflicted with this genetic disorder. This disease is not limited to Tervuren population in the USA.

How is the DNA collected?

DNA is collected from cells obtained by buccal swabs (cheek cells that you collect on a special "Q-tip" rolled on the inside of a dog?s cheek)

Can you help?

YES!

Contact Dr. Anita Oberbauer via email below.

Confidentiality

We realize that this is a sensitive topic and some of you may be willing to participate, but only under confidentiality. We have arranged for that also. The previous ABTC survey was collected with the understanding of confidentiality. That goal was achieved in the survey analyses presented here. Computer programmers were screened for their lack of association with purebred dogs. These programmers input the names, pedigrees, and seizure information into a large database. Then the dog names were encrypted such that individual names were replaced with numbers. The analyses for the previous data, as well as what we propose here, only relies upon pedigree relationships; names are irrelevant to the success of the genetic marker search.

Remaining Anonymous

If you are interested in participating in the study, but wish to have the dogs remain anonymous, write or E-mail Dr. Thomas Famula.  He will send out DNA collection kits which include directions and diagrams for collecting, a return envelope, and questionaire for pedigree and seizure information.

When the samples are returned, the dog?s identity will be encrypted and a numerical identification assigned. That number will be ascribed to that dog?s DNA. The dog?s identity will remain anonymous, yet the pedigree associations will remain intact for the study.

Contacts

A genetic marker linked to this major gene will significantly contribute to minimizing seizures in our breed. We believe such a search will yield a marker.

If you have any questions regarding any part of this study, please feel free to contact either Dr. Famula or Dr. Oberbauer.

Dr. Thomas R. Famula Dr. Anita M. Oberbauer
rfamula@ucdavis.edu amoberbauer@ucdavis.edu
Dept of Animal Science Dept of Animal Science
University of California University of California
Davis, CA 95616 Davis, CA 95616

References:

Famula, T.R., Oberbauer, A.M., Brown, K.N. 1997. Heritability of epileptic seizures in the Belgian Tervuren. J. Small Animal Practice 38:349-352.

Famula, T.R., Oberbauer, A.M. 1998. Reducing the incidence of epileptic seizures in the Belgian Tervuren through selection. Preventative Veterinary Medicine 33:251-159.

AKC Grant Information

Grant: Development of a Genetic Marker for Idiopathic Epilepsy in the Belgian Tervuren

Principal
Investigator
Co-Principal
Investigators
Co-Principal
Investigators
A.M. Oberbauer, PhD. T. R. Famula, PhD B. May, PhD.
University of CA, Davis University of CA, Davis University of CA, Davis
Dept. of Animal Science Dept. of Animal Science Genomic Variation Laboratory
Davis, CA 95616 Davis, CA 95616 Davis, CA 95616

This document is designed to introduce the scientific reasonings behind the grant given to the Department of Animal Sciences at the University of California, Davis.

Abstract

Idiopathic epilepsy affects nearly 30 different dog breeds.

Preliminary studies in the Belgian Tervuren indicates that although this is a heritable disorder governed by many genes (polygenic mode of inheritance), there exists a single gene of very large effect on the incidence of epilepsy. The epileptic condition influenced by this particular gene is inherited as an autosomal recessive.

The specific objective of this project is to develop a genetic marker associated with that locus in the Belgian Tervuren by utilizing the emerging canine genetic map.

Such a genetic marker will permit the identification of carriers of epilepsy in Belgian Tervuren, and possibly other breeds as well, with the end result that breeders can make informed, health-based breeding decisions.

Significance of Research

Seizures have been reported in nearly all dog breeds; where a seizure is defined as "a transitory disturbance of brain function" (2). Often, the seizure reflects an isolated incident and does not recur.

Recurrent seizure activity defines epilepsy (16) with the epilepsy categorized by its causality. Idiopathic or primary epilepsy (Idiopathic Epilepsy) describes recurrent seizures with no discernible cause. Acquired or secondary epilepsy is identifiable by a specific initiator of the seizure, such as trauma or hepatic insufficiency (9).

Breeds Affected by Epilepsy

Epilepsy accounts for 0.55% to 2.3% of all ill dogs referred to veterinary teaching hospitals (20). A growing body of evidence supports a hereditary basis for Idiopathic Epilepsy (7,12,13,16). Many AKC recognized breeds are suspected of having a predisposition to Idiopathic Epilepsy. These breeds (compiled from 4,16) include:

Petit Basset Griffon Vendeen Dachshund
Golden Retriever Bichon Frise
German Shepherd Irish Setter
Italian Greyhound Ibizan hound
Labrador Retriever Beagle
English Toy Spaniel Brittany
American Cocker Spaniel Collie
Fox Terrier Finnish Spitz
St. Bernard Canaan dog
Keeshond Poodle
Flat Coated Retriever Pointer
Alaskan Malamute Boxer
Siberian Husky Corgi
Gordon Setter

In addition, Idiopathic Epilepsy has long been recognized in the Belgian Tervuren (25), a breed with a 17% seizure incidence in the U.S. (7).

With such prevalence among dog breeds and high incidence within certain breeds, a genetic marker identifying deleterious alleles in breeding stock is extremely desirable.

And while prevalence among breeds generally reflects heterogeneity in the allelic causation, it has been demonstrated with other disorders, such as von Willebrand's disease (1), that within a breed the allelic mutation is homogeneous.

The high incidence of Idiopathic Epilepsy in the Belgian Tervuren, coupled with the cooperation and willingness of Belgian Tervuren breeders to eliminate this disorder, translates into an optimal population to initiate a study for a genetic marker linked with Idiopathic Epilepsy. Linkage of Idiopathic Epilepsy with such a marker and the subsequent identification of the affected gene will allow the information to be applied to other breeds also afflicted with Idiopathic Epilepsy.

Background of Research and Preliminary Work

Idiopathic Epilepsy, reported in nearly thirty different dog breeds (4,16), is considered a hereditary disorder (5), especially if the onset of the initial seizure is between 1-5 years of age (15).

A variety of genetic models have been proposed for the inheritance of canine Idiopathic Epilepsy, drawing primarily on human and mouse models of epilepsy. In humans, many of the characterized epileptic syndromes involve single locus modes of inheritance (11,27) though canine models have been consistent with multilocus modes (7,24) excepting the single locus model proposed for Idiopathic Epilepsy in the Keeshond (12).

Belgian Tervuren breeders worldwide have long recognized the necessity of determining the transmission of Idiopathic Epilepsy in the breed (25) due to its high frequency; understandably with a high incidence, a genetic basis was presumed. Although early studies identified a genetic component (25), the mode of inheritance remained elusive. In late 1986, the American Belgian Tervuren Club initiated a confidential survey of its members as to the incidence of seizures in Belgian Tervuren: all members were surveyed and 91% returned completed questionnaires (6).

From this collected data, 997 unique Tervuren over the age of five years of age were analyzed. This data revealed:

  • 17% incidence of Idiopathic Epilepsy
  • no significant difference in seizuring across sexes
  • heritability estimate of 0.77 for Idiopathic Epilepsy using a Bayesian analysis (7); an even higher estimate was obtained with Gibbs sampling

This estimate suggests, congruent with previous reports of Idiopathic Epilepsy in other breeds (22,24), that Idiopathic Epilepsy is a highly heritable disorder in the Belgian Tervuren.

The high heritability estimates confirm that there is sufficient genetic variation in the incidence of Idiopathic Epilepsy to guarantee an effective response to selective breeding and implementation of sound selection decisions. Heritability estimates in excess of 0.5 indicate the presence of a major gene (18).

Although the evidence in the Tervuren suggests that a single locus model is not plausible, multiple statistical tests applied to the data set presented results consistent with a mode of inheritance involving a single autosomal gene of large effect that influences the expression of seizures in the Belgian Tervuren (8).

The statistical tests employed to reach this conclusion included:

  • test of global misture distributions for predicted breedgin values
  • tests for heterogeneity, polynomial regression and structured exploratory data analysis 

Polygenic inheritance with a single gene of large effect is consistent with epileptic seizure activity in the dog: seizure potential has been characterized as a continuum of susceptibility(9). Such a continuum could represent many genes contributing to a seizure threshold with a single locus exerting great influence on that threshold.

Specific Objectives of the Study

Statistical evidence of a single locus of large effect in a polygenic trait has been exploited in other species to develop genetic markers to assist in selection of particular breeding stock (23). We plan on implementing a similar strategy for the development of a genetic marker useful in identifying carriers of deleterious alleles predisposing offspring to Idiopathic Epilepsy. The high estimate of heritability, coupled with the existence of appropriate family pedigrees, argues for likely success in this endeavor.

Our specific aim for this two year proposal is to find a microsatellite genetic marker linked to Idiopathic Epilepsy in the Belgian Tervuren.

Expected Outcomes and Application to All Dogs

We will utilize the canine genome map that is being developed which is coupled with the ever expanding polymorphic canine microsatellites. Our preliminary data supports that we have a high likelihood of developing such a marker and this will be invaluable to breeders of Belgian Tervuren.

Our long term research objective is to identify and sequence the major gene involved in the Tervuren Idiopathic Epilepsy. This objective is in conjunction with the goal of determining if the other breeds afflicted with Idiopathic Epilepsy will show similar or related mutations.

As ascertained by the annual survey of AKC Parent Club Health Representatives, epilepsy is a major concern to dog breeders.

Identification of a genetic marker and the high heritability of Idiopathic Epilepsy in the various breeds of dogs would allow a significant reduction in the incidence of this debilitating disorder and ultimately improve the health status of the pure-bred dog.

References

 

  • Brewer G. 1997. Summary of Dr. Brewer's opening remarks to epilepsy session. AKC Canine Health Foundation: Molecular Genetics and Canine Genetic Health Conference proceedings, page 27.
  • DeLahunta A. 1977. Veterinary neuroanatomy and clinical neurology. WB Saunders Co., Philadelphia.
  • Delgado-Escueta AV, Serratose JM, Liu A, Weissbecker, K, Medina MT, Gee M, Treiman LJ, Sparkes RS. 1994. Progress in mapping human epilepsy genes. Epilepsia 35(suppl 1): S29.
  • Clark RD, Stainer JR. 1994. Medical & genetic aspects of purebred dogs. Forum Publications, Inc. Georgia
  • Cunningham JG, Farnbach GC. 1988. Inheritance of idopathic canine epilepsy. J Am Anim Hosp Assoc
  • Famula TR, Oberbauer AM. 1997. Reducing the incidence of epileptic seizures in the Belgian Tervuren through selection. Prev Vet Med 1183:in press.
  • Famula TR, Oberbauer AM, Brown KN. 1997. Heritability of epileptic seizures in the Belgian Tervuren. J Sm Anim Prac 38:349
  • Famula TR, Oberbauer AM. 1997. Statistical evidence for a single gene of large effect on epilepsy in the Belgian Tervuren. AKC Canine Health Foundation: Molecular Genetics and Canine Genetic Health Conference proceedings, page 107.
  • Farnbach GC. 1984. Seizures in the dog: Part I. Basis, classification, and predilection. Comp Cont Educ 6:569
  • Francisco LV, Langston AA, Mellersh CS, Neal CL, Ostrander EA. 1996. A class of highly polymorphic tetranucleotide repeats for canine genetic mapping. Mam Genome 7:359
  • Greenberg DA, Durner M, Resor S, Rosenbaum D, Shinnar S. 1995. The genetics of idiopathic epilepsies of adolescent onset. Neurology 45:942
  • Hall Sj, Wallace ME. 1996. Canine epilepsy: a genetic counselling programme for keeshonds. Vet Rec 138:158
  • Heynold Y, Faissler D, Steffen F, Jaggy A. 1997. Clinical, epidemiological and treatment results of idiopathic epilepsy in 54 labrador retrievers: a long term study. J Sm Anim Prac 38:7
  • Kapfhamer D, Sweet HO, Suflako D, Warren S, Johnson KR, Burmeister M. 1996. The neurological mouse mutations jittery and hesitant are allelic and map to the region of mouse chromonsome 10 homologous to 19p13.3. Genomics 35:533
  • Knowles K. 1994. Seizure disorders in the pediatric animal patient. Sem Vet Med Surg 9:108
  • LeCouteur RA, Child G. 1989. Clinical management of epilepsy in dogs and cats. Prob Vet Med 1:578
  • May B, Gavin TA, Sherman PW, Korves TM. 1997. Characterization of microsatellite loci in the Northern Idaho ground squirrel Spermophilus brunneus brunneus. Mol Ecol 6:399
  • Morton NE, MacLean CJ. 1974. Analysis of family resemblance. III. Complex segregation of quantitative traits. Amer J Hum Genet 26:489
  • Ostrander EA, Mapa FA, Yee M, Rine J. 1995. One hundred and one new simple sequence repeat- based markers for the canine genome. Mam Genome 6:192
  • Podell M, Fenner WR. 1993. Bromide therapy in refractory canine idiopathic epilepsy. J Vet Int Med 7:318
  • Richards B, Skolestky J, Shuber AP, Balfour R, and others. 1993. Multiplex PCR amplification form the CFTR gene using DNA prepared from buccal brushes/swabs. Hum Mol Genet 2:159
  • Schwarts-Porsche D. 1994. Seizures. In: Clinical Syndromes of Veterinary Neurology. KG Braund, ed. Mosby-Year Book, St. Louis. pp234
  • Spelman RJ, Coppieters W., Karim L, van Arendonk JA, Bovenhuis H. Quantitative triat loci analysis for five milk production traits in the Dutch Holstein-Friesian population. Genetics 1996. 144:1799
  • SrenkP, Gaillard A, Busato C, Horin P. 1994. Genetische grundlagen der idiopathischen epilepsie biem Golden Retriever. Teirarztliche Praxis 22:574
  • Van der Velden NA. 1968. Fits in Tervuren shepherd dogs: a presumed hereditary trait. J Sm Anim Prac 9:63
  • Mellersh CS, Langston AA, Acland GM, Fleming MA, Ray K, Wiegand NA,
    Francisco LV, Gibbs M, Aguirre GD, Ostrander EA. 1997. A linkage map of
    the canine genome. Genomics 46:326-336

 

Clinical and Genetic Advances

Inherited canine epilepsy is a major health problem in many breeds because of its high frequency and it potentially serious effects on pet ownership and breeder reputation. A preliminary examination of published data indicate that over 20 breeds have a serious health problem with canine epilepsy.

Read more: Clinical and Genetic Advances

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