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: john.angles@bigpond.com

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.

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.

BACKGROUND OF 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).

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.

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.

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 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.

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.

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. J Nutr, 129: 1966-1971

6.Dougherty, SA, Center, SA, Shaw, EE and Erb, HA (1991) Juvenile-onset polyarthritis syndrome in Akitas. JAVMA, 198 (5): 849-856

7.Goodman, SA, Montgomery, RD, Fitch, RB, Hathcock, JT, et al (1998) Serial Orthopedic Examinations of Growing Great Dane Puppies Fed Three Diets Varying in Calcium and Phosphorus. In Recent Advances in Canine and Feline Nutrition, volume II. Reinhart, GA and Carey, DP. (Eds) Iams Nutritional Symposium proceedings. Pp. 3-12

8.Grondalen, J (1976) Metaphyseal osteopathy (hypertrophic osteodystrophy) in growing dogs. A clinical study. JSAP, 17: 721-735

9.Holmes, JR (1962) Suspected skeletal scurvy in the dog. Vet Rec, 74: 801-813

10. Malik, R, Dowden, M, Davis, PE, Allan, GS, Barrs, VR, Canfield, PJ and Love, DN. (1995) Concurrent Juvenile Cellulitis and Metaphyseal Osteopathy: An Atypical Canine Distemper Virus Syndrome? Aust Vet Pract, 25 (2): 62-67

11. Meier, H, Clark, ST, Schnelle, GB, Will, DH (1957) Hypertrophic osteodystrophy associated with disturbance of vitamin C synthesis in dogs. JAVMA, 130: 483-494

12. Mee, AP, Gordon, MT, May, C, Bennett, D, Anderson, DC and Sharpe, PT (1992) Canine Distemper Virus Transcripts Detected in the Bone Cells of Dogs with Metaphyseal Osteopathy. Bone, 14: 59-67

13. Mee, AP, Webber, DM, May, C, Bennett, D, Sharpe, PT and Anderson, DC (1993) Detection of Canine Distemper Virus in Bone Cells in the Metaphyses of Distemper-infected Dogs. J Bone and Mineral Research, 7 (7): 829-834

14. Muir, P, Dubielzig, RR, Johnson, KA, Shelton, GD (1996) Hypertrophic osteodystrophy and calvarial hyperostosis. CCEPV, 18(2): 143-151

15. Munjar, TA, Austin, CC, and Breur, GJ (1998) Comparison of Risk Factors for Hypertrophic Osteodystrophy, Craniomandibular Osteopathy and Canine Distemper Virus Infection. Vet Comp Orthop Traumatol, 11: 37-43

16. Parker, WM and Foster, RA (1996) Cutaneous vasculitis in five Jack Russell Terriers. Vet Dermatology, 7: 109-115

17. Riser, WH, Shirer, JF (1965) Normal and abnormal growth of the distal foreleg in large and giant dogs. J Am Vet Radiol Soc, 6: 50-64

18. Scott-Moncrieff, JCR, Snyder, PW, Glickman, LT, Davis, EL and Felsburg, PJ (1992) Systemic necrotizing vasculitis in nine young Beagles. JAVMA, 201 (10): 1553-1558

19. Snyder, PW, Kazacos, EA, Scott-Moncrieff, JC, HogenEsch, H, Carlton, WW, Glickman, LT and Felsburg, PJ (1995) Pathological Features of naturally Occurring Juvenile Polyarteritis in Beagle Dogs. Vet Pathol, 32: 337-345

20. Sorensen, D. A., Andersen, S., Gianola, D., and Korsgaard, I. 1995. Bayesian inference in threshold models using Gibbs sampling. Genetique, Seletion, Evolution. 27:229-249

21. Woodard, JC (1982) Canine hypertrophic osteodystrophy, a study of the spontaneous disease in littermates. Vet Pathol, 19: 337-354 "--------------------------------------------------------------------------------

This material is copyright April, 16th 2001 by Dr. John M. Angles. No portion of this text may be reproduced in any form or distributed by any method, whether for profit or not, without the express written permission of Dr. John M. Angles.