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: <john.angles@ucd.ie>
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:
2000
Diplomate
Companion Animal Medicine, ECVIM
1998
Diplomate
Small Animal Medicine, ACVIM
1993
MVStudies Murdoch University, Australia
1993
MACVSc
Canine Medicine
1988
BVSc
Honors Class 1, University of Sydney, Australia
1987
BSc (Vet)
Veterinary Physiology, University of Sydney, Australia
Honors, Awards:
1999
Phi Sigma Society, Gamma Delta Society
1996-1997 Clinical Research Fellowship in Immunogenetics, University of California, Davis
1988
Martin McIllwraith Scholarship, University of Sydney, Australia
1986
Selby Scientific Foundation Scholarship in Ultrasonography, University of Sydney
1984
Harrie 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
1996-1999 Postdoctoral 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. 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