TITLE: “IDENTIFICATION OF A GENETIC MARKER FOR HYPERTROPHIC OSTEODYSTROPHY IN THE WEIMARANER”

PRINCIPAL INVESTIGATOR:

Dr. John M. Angles

BVSc, BSc (Vet), DACVIM, DECVIM-CA

Ralston Purina Lecturer in Genetics and Medicine

Faculty of Veterinary Medicine

University College Dublin

Ballsbridge, Co Dublin 4 IRELAND

Phone: +353-1-668-7988, extension 2632

Fax: +353-1-667-5401

Email: <>

COLLABORATORS:

Dr. Niels C. Pedersen, DVM, PhD

Director, Veterinary Genetics Laboratory

University of California, Davis

Dr. Thomas Famula, PhD

Professor, Department of Animal Science

University of California, Davis

2) TABLE OF CONTENTS:

Science Summary

Lay Abstract

Biographical Sketch of Principal Investigator

John M. Angles

Current Funding Sources

Facilities & Research Environment

Preliminary Work

Research Plan

Specific objectives

Background of research

Expected outcome, significance and application of findings

Research design and methods

Schedule of work to be performed

Animals and institutional animal care and use statement

Acknowledgment

3) SCIENCE SUMMARY

Hypertrophic osteodystrophy (HOD) is a common disease of rapidly growing dogs of the large and giant pure-breeds. Breeds found to be at higher risk for HOD include the Great Dane, Weimaraner, Irish Setter and German Shepherd. The disease in the Weimaraner is particularly severe, with high mortality rates found in untreated dogs. The age of onset is typically 8 to 16 weeks of age, with males and females equally affected. Recent vaccination with multivalent vaccines (<7 days) is a significant risk factor for occurrence of disease. Genetic susceptibility to HOD is indicated in the Weimaraner, with a heritability of 0.35 and an autosomal recessive mode of inheritance. Unfortunately, to date there is no method available to detect carriers other than by test matings and this approach has been ineffective in controlling the disease. Ten informative 3-4 generation families are available for linkage analysis using the minimum mapping set of 172 microsatellite markers. Determination of a genetic marker for susceptibility to HOD in the Weimaraner will have immediate impact in the breed for genetic counselling. An understanding on the pathogenesis of HOD and hyper-inflammatory diseases in general may have broad implications for other breeds with these diseases.

4) LAY ABSTRACT

Hypertrophic Osteodystrophy (HOD) is a common disease of the rapidly growing dogs of the large and giant pure-breeds. Breeds found to be at higher risk for HOD include the Great Dane, Weimaraner, Irish Setter and German Shepherd. The disease in the Weimaraner is particularly severe, with high mortality rates found in untreated dogs. The age of onset is typically 8 to 16 weeks of age, with males and females equally affected. Recent vaccination with multivalent vaccines (<7 days) is a significant risk factor for occurrence of disease. HOD in the Weimaraner is a genetic disease, with a heritability of 0.35 and an autosomal recessive mode of inheritance. Unfortunately, to date there is no method available to detect carriers other than by test matings and this approach has been ineffective in controlling the disease. DNA samples from 10 separate 3-4 generation families with HOD have been collected, and we propose to identify a marker for susceptibility to HOD in the Weimaraner breed. Determination of a genetic marker for HOD in the Weimaraner will have immediate impact in the breed for genetic counselling. An understanding on the pathogenesis of HOD and hyper-inflammatory diseases in general may have broad implications for other breeds with these diseases.

5) BIOGRAPHICAL SKETCH of PRINCIPAL INVESTIGATOR

JOHN M. ANGLES:

Education:

2000DiplomateCompanion Animal Medicine, ECVIM

1998DiplomateSmall Animal Medicine, ACVIM

1993 MVStudies Murdoch University, Australia

1993 MACVScCanine Medicine

1988 BVScHonors Class 1, University of Sydney, Australia

1987BSc (Vet)Veterinary Physiology, University of Sydney, Australia

Honors, Awards:

1999Phi Sigma Society, Gamma Delta Society

1996-1997 Clinical Research Fellowship in Immunogenetics, University of California, Davis

1988Martin McIllwraith Scholarship, University of Sydney, Australia

1986 Selby Scientific Foundation Scholarship in Ultrasonography, University of Sydney

1984Harrie Barrett Bursary, University of Sydney, Australia

Research and Professional Experience:

1999-Pres.Ralston Purina Lecturer in Small Animal Medicine and Genetics, Faculty of Veterinary

Medicine, University College Dublin, Ireland

1998- Pres.Member of the ISAG DLA Nomenclature Committee

1996-1999Postdoctoral Researcher, University of California, Davis

1994-1996 Resident, Small Animal Medicine, Veterinary Medical Teaching Hospital,

University of California, Davis

Publications:

1)Angles, JM and Pedersen, NC (2001) Role for the MHC in Uveodermatological syndrome (VKH-like) disease in the Akita dog. Tissue Antigens (submitted)

2)Angles, JM, Kennedy, LJ, Haap, GM, Barnes, A, and Ollier, WER (2001) Geographical variation of MHC alleles and haplotypes in Alaskan Grey Wolves. Animal Genetics (submitted)

3)Kennedy, LJ, Altet, L, Angles, JM et al. Nomenclature for the DLA system, 1998. (2000) First report of the ISAG DLA Nomenclature Committee. Tissue Antigens, 54: 312-321

4)Abeles, V, Harrus, S, Angles, JM, Shalev, G, Aizenberg, I, Peres, Y and Aroch, I (2000) Hypertrophic osteodystrophy in six Weimaraner puppies associated with systemic signs. Vet Record, 145: 130-134

5)Angles JM, Feldman EC, Nelson RW and Feldman MS (1997) Use of Urine Cortisol: Creatinine Ratio versus Adrenocorticotrophic Hormone Stimulation Testing for Monitoring Mitotane Treatment of Pituitary-dependent Hyperadrenocorticism, JAVMA 211 (8): 1002-1004

6)Angles JM, Walsh DA, Li K, Barnett SB and Edwards MJ (1990) The Effect of Temperature and Pulsed Ultrasound on the Development of Rat Embryos in Culture, Teratology 42: 285-293

Specific role:

Primary responsibility for co-ordination of this international collaboration, with DNA samples being distributed from the Weimaraner DNA/disease database held at University College Dublin. Minimum mapping set to be run here at UCD. Publication of scientific results. Direct consultation with the Weimaraner Club of America regarding genetic counselling and use of potential DNA marker.
6) CURRENT FUNDING SOURCES:

The maintenance of the Weimaraner DNA-disease database and continued development of the pedigrees for linkage analysis is currently supported by a generous gift from the Yankee Weimaraner Club. This funding will finish in July 2001.

7) FACILITIES AND RESEARCH ENVIRONMENT:

This research will be conducted at the laboratories of the Molecular Biology Unit at University College Dublin and the Veterinary Genetics Lab at UCDavis.

The Molecular Biology Unit at University College Dublin is a primary research facility within the Veterinary School. Research space and equipment are shared across the Faculty, with fume and bio-hoods, incubators, centrifuges, thermocyclers, electrophoresis devices, refrigerators, freezers, computers, and other equipment for biomedical research available at all times. The Wellcome Foundation (UK) has recently installed a 377 DNA automated sequencer for use by researchers in the Veterinary Faculty.

The Veterinary Genetics Laboratory is a production and research facility working on genetic typing tests for the canines, equine, bovine, as well as other exotic stock species. In addition to an experienced technical staff, the Veterinary Genetics Laboratory also has made available two ABI 377 automated sequencing machines for use by researchers in the School of Veterinary Medicine in genetic typing or gene sequencing research.

8) PRELIMINARY STUDIES:

The Weimaraner is at high risk for HOD disease compared to most breeds. Using the Veterinary Medical Database (VMDB) at Purdue, the Weimaraner is 16.4 times more likely to develop HOD compared to the pooled population (Angles, unpublished data). Other breeds at high risk included the Great Dane (23.1), Irish Setter (10.4) and Irish Wolfhound (11.4). To date, the Weimaraner DNA-disease database contains information for 70 Weimaraners that have been diagnosed with HOD. Criteria for diagnosis of HOD are strict, and include presence of appropriate clinical signs (swollen painful metaphyses; fever; lameness), radiographic evidence of HOD, and exclusion of other “look alike” diseases. Males and females are equally affected, and the age of onset is typically 8 to 16 weeks of age (Angles and Pedersen, 2001).

An important finding of our studies has been the association of the HOD disease with recent vaccination. In our cohort of Weimaraners in North American, 80% of the dogs diagnosed with HOD had received a multivalent vaccination within 7 days of disease onset (most within 2 to 3 days). The vaccine is probably not the cause for disease but acts as a trigger for expression of HOD disease on a susceptible genetic background. We are currently examining the role of the major histocompatibility complex (MHC) and cytokine expression in the pathogenesis of HOD disease, but consider these to be modifiers of disease expression rather than the major gene causing HOD.

The genetics of Hypertrophic osteodystrophy (HOD) in the Weimaraner have been studied in AKC (CHF) grant # 1628. The mode of inheritance of HOD in the Weimaraner is autosomal recessive based on retrospective analysis of over 80 HOD affected Weimaraners and their extended families (Angles and Famula, 2001). Based on a conservative estimate of 2.8% for the prevalence of HOD in the Weimaraner (Angles, unpublished data), some 30% of the breed are believed to be carriers for this debilitating disease. DNA samples for 70 HOD affected Weimaraners are available, and pedigree analyses have identified ten 3-4 generation informative families segregating HOD disease. DNA samples are stored at the University College Dublin and are freely available for collaboration with other investigators by petition to the principal investigator.

In preparation for linkage analysis, 100 microsatellite markers available on the UCDavis multiplexed linkage sets were assessed over 100 unrelated Weimaraners. These linkage sets have 14 individual markers per set and have been designed to give 15-20cM coverage of the canine chromosomes. Results of this analysis were encouraging, as this population had been considered highly inbred prior to DNA analysis. Most markers had PIC values between 0.50 and 0.85 (available on request), but several markers performed poorly in these dogs (AHT132, CO3.877, CPH03, CPH16, FH2293, FH2361, LEI005, LEI007, Wilms-TF). Markers AHT136, AHTk211, AHTk292, CO1.424, CO6.636, CO9.173, CO9.250, CXX391, CXX608, FH2004, FH2148, FH2328, FH2356, LEI004, LEI006, PEZ02, PEZ05, RVC1, and VIASD10 had PIC values <0.3, which may limit their usefulness for a subsequent linkage study. It is proposed to supplement the UCDavis multiplex linkage set with markers available on the Research Genetics minimum mapping set to give 15cM preliminary chromosomal coverage.

9) RESEARCH PLAN:

SPECIFIC OBJECTIVES OF THE STUDY:

Our primary objective is to identify a DNA marker for hypertrophic osteodystrophy (HOD) in the Weimaraner breed. Locating a genetic marker for HOD in the Weimaraner will have immediate impact in the breed for genetic counselling.

Further, knowledge of the physical location of the marker/s will facilitate studies examining the actual cause for this disease. An understanding on the pathogenesis of HOD and hyper-inflammatory diseases in general may have broad implications for purebred dogs.

BACKGROUND OF PROPOSED RESEARCH:

Hypertrophic Osteodystrophy (HOD) is a common disease of rapidly growing, large to giant pure-breeds of dog. Most dogs present with pain and lameness associated with swelling of the metaphyses of the long bones. Fever and anorexia are also commonly associated with clinical HOD (Muir, 1996). The Weimaraner breed unfortunately has a severe form of HOD disease compared to most breeds, with systemic manifestations including fever and multiple body organ inflammation (Abeles, 1999; Angles and Pedersen, 2001). Diagnosis of HOD relies on the typical signalment, clinical signs, and presence of pathognomic radiographic changes in the metaphyses.

The cause of canine HOD remains unknown, with earlier speculations of a vitamin C deficiency (Meier, 1957; Holmes, 1962) or over-nutrition (Riser, 1965) largely discounted in more recent times (Grondalen, 1976). Expression of HOD disease has been linked to calcium content of the diet, with Great Dane pups fed on a high calcium diet showing HOD and those on a low calcium diet not showing disease (Goodman, 1998). However, Great Dane puppies from susceptible lines fed on restricted calcium diets still show signs of HOD disease, albeit with lesser severity (Great Dane Club of America, personal communication). This suggests that calcium levels influence expression of the inflammatory component of the disease but are not directly causative for the disease. A similar effect has been seen in experimental autoimmune encephalomyelitis (EAE) in mice, where low dietary calcium led to protection against EAE (Cantorna, 1999).

There is mounting evidence that viral infection may be important in the disease, with Distemper virus detected in the metaphyses of dogs with HOD (Mee, 1992; Mee, 1993; Baumgartner, 1995). Several authors (Abeles, 1999; Malik, 1995) have also noted a temporal association with Distemper vaccination and onset of HOD disease. However, to our knowledge no one has been able to experimentally reproduce HOD with Distemper Virus infection, suggesting that there are other important factors leading to expression of this disease. In such a scenario, the Distemper virus would act as a trigger for the initial immune response but expression of the disease would depend on other immune regulatory factors.

The Weimaraner dog is heavily represented in case reports of HOD in the veterinary literature, with documented familial clustering (Grondalen, 1976; Woodard, 1982; Abeles, 1999). In a recent study looking at breed predilection for developmental orthopedic diseases, the Weimaraner was found to be 21 times more likely to develop HOD compared to mixed breed dogs (Munjar, 1998). Similarly, the Great Dane (190x), the Boxer (18.4x), the Irish Setter (14.3x), and the German Shepherd (9.5x), were at increased risk for HOD. Our work has confirmed that HOD in the Weimaraner is a genetic disease, with a heritability of 0.35 and an autosomal recessive mode of inheritance (Angles and Famula, 2001).

EXPECTED OUTCOME, SIGNIFICANCE AND APPLICATION OF RESEARCH:

Our primary objective is to identify a DNA marker for hypertrophic osteodystrophy (HOD) in the Weimaraner breed. Locating a genetic marker for HOD in the Weimaraner will have immediate impact in the breed for genetic counselling. For the first time, breeders will be able to perform selective breeding to decrease the prevalence of carriers and thereby disease in the breed. Twenty years of selection based on test matings has unfortunately not been able to achieve this objective.

Detection of a DNA marker for HOD in one breed will be critical for furthering our knowledge on the pathogenesis of HOD disease. Knowledge of the physical location of the DNA marker on the canine chromosome may permit rapid identification of a candidate gene for HOD disease susceptibility across many breeds. The Weimaraner breed will give us the potential to dissect complex environmental influences from underlying genetic risk factors to determine if there is a common cause for HOD in the dog. The benefits for all pure-breeds of dog will significant.

RESEARCH DESIGN:

DNA samples for the ten informative families are already available for marker analysis at the University College Dublin. Based on pedigree and preliminary marker analysis, we believe that a classical linkage study will be the appropriate approach to analyzing these families.

The minimum mapping set of 172 microsatellite markers available through Research Genetics will be used to attain 15cM coverage of the chromosomes to look for linkage. Some of the markers available in the minimum mapping set are already available in multiplex sets of markers at UCDavis. Preliminary data on these multiplex sets suggests that some 25 markers will need replacement to ensure even chromosome coverage for the Weimaraner breed.

Markers available in the multiplex panels will be run at the Veterinary Genetics Laboratory at UCDavis. University College Dublin will be responsible for performing analysis for markers in the minimum mapping set not available through UCDavis. Markers added to replace gaps in the chromosome coverage would be validated and run at University College Dublin. Both facilities have in-house ABI 377 DNA sequencers and full range of molecular biology equipment.

Markers will initially be run across three pools of samples (i.e. known affected, known carriers and “probable” clears) looking for areas homozygous in known affected and heterozygous in known carrier animals. This will allow us to increase the marker saturation at an early stage in areas of potential interest. Marker analysis will then be completed in the linkage families. In all families, a preliminary panel of 15 microsatellites will be run for confirmation of the pedigrees as supplied.

Statistical analysis will be performed using algorithms designed by Dr. Tom Famula at the UCDavis. These algorithms integrate previous pedigree information available on the DNA-disease database at University College Dublin with the allele information as determined by both labs. These techniques have already been validated using for linkage analysis for seizures in the Belgian Tervuren.

SCHEDULE OF WORK TO BE PERFORMED:

1)DNA samples are already available for marker analysis. Pedigrees for linkage analysis are available on request to principal investigator.

2)Markers not already available through the UCDavis multiplex linkage sets will be purchased from Research Genetics at the beginning of the grant.

3)Preliminary analysis of “pooled” samples and paternity testing of linkage families within 2 month of start of grant. Validation of new markers during this period (PIC, Htz).

4)Complete marker allele acquisition is anticipated to be complete at the end of the eighth month of the grant. Completed data set forwarded to Dr. Famula for analysis.

5)Completed linkage analysis by the end of the tenth month of the grant.

6)Scientific and breed-club publications at the end.

ANIMAL AND INSTITUTIONAL LABORATORY ANIMAL CARE STATEMENT:

DNA in the Weimaraner DNA-disease database at University College Dublin is derived from buccal swabs and/or whole blood taken with the full cooperation of owners, breeders, and veterinarians. No invasive animal experiments will be done. No laboratory animals are being kept for this research.

LITERATURE CITED:

  1. Abeles, V, Harrus, S, Angles, JM, Shalev, G, Aizenberg, I, Peres, Y and Aroch, I (1999) Hypertrophic osteodystrophy in six Weimaraner puppies associated with systemic signs. Vet Record, 145: 130-134
  2. Angles, JM and Pedersen, NC (2001) Hypertrophic Osteodystrophy (HOD) in the Weimaraner: 60 cases. JAAHA (in preparation)
  3. Angles, JM and Famula, TR (2001) Genetics of Hypertrophic Osteodystrophy (HOD) in the Weimaraner. JVIM (in preparation)
  4. Baumgartner, W, Boyce, RW, Alldinger, S, Axthelm, MK, Weisbrode, SE, Krakowka, S and Gaedke, K (1995) Metaphyseal bone lesions in young dogs with systemic distemper virus infection. Vet Microbiol, 44: 201-209
  5. Cantorna, MT, Humpal-Winter, J, and DeLuca, HF. (1999) Dietary Calcium is a Major Factor in 1, 25-Dihydroxychlecalciferol Suppression of Experimental Autoimmune Encephalomyelitis in Mice.