Heritability & Transmission Analysis of Pug Dog Encephalitis
ACVIM 2008
Kimberly A. Greer, PhD; Scott J. Schatzberg, DVM, DACVIM, PhD; Brian F. Porter, DVM, DACVP; Kim A. Jones, BS; Thomas R. Famula, PhD; Keith E. Murphy, PhD
College Station, TX, USA

Introduction

Necrotizing meningoencephalitis is an idiopathic inflammatory disease of the central nervous system that is unique to several breeds of small-sized dogs. The best characterized and perhaps most common form of the disease is Pug Dog Encephalitis (PDE), which was first described in California twenty years ago1 and has since been documented elsewhere in the United States and in several other countries.2,3,4,5 PDE typically affects young adult Pugs and clinical signs include lethargy, depression, seizures, proprioceptive deficits, circling, and blindness. The disease progresses rapidly, and usually within days or weeks of the first clinical signs, dogs culminate in status epilepticus or coma6,7,8. The average age of death in affected dogs is 16 months, but ranges from 6 months to 7 years of age. Immunosuppressive and anticonvulsant drugs have allowed some survival for up to seven months after onset, but long term recovery has not been described.4,7,8,9 The pathologic findings are distinctive, consisting of inflammation and necrosis largely focused on the cerebral hemispheres.1,3,10

The cause of PDE is unknown. Previous reports speculated that a virus, perhaps canine herpesvirus, may play a role in the pathogenesis6; however, attempts at proving viral etiology have been unsuccessful.11 The breed predilection strongly suggests a genetic component.1,3,7,8,12,13 In the original report, eleven of seventeen affected dogs originated from one kennel and had numerous common ancestors.1 Affected dogs may have increased susceptibility to an infectious agent or an inappropriate immune response to an infectious agent or self-antigen. Understanding the genetic component(s) of PDE is a requirement for the development of tests to identify carriers of the allele(s) contributing to the disease. Such tests could be used to eliminate or reduce the incidence of the disease through judicious breeding practices. Data supporting direct transmission are nonexistent, so the current study was undertaken to begin comprehensive analyses of the heritable component(s) involved in PDE.

Material and Methods

Animals

Study participants were recruited with cooperation from the Pug Dog Club of America, interested breeders, and individual Pug owners. Study protocols were approved by the animal care and use committee of Texas A&M University (TAMU). Post mortem histopathology of brain tissue to distinguish PDE from granulomatous meningoencephalitis (GME), was required. Buccal brushes for epithelial cell DNA extraction were solicited from living relatives of each deceased Pug, and pedigree records requested. Complete medical records for Pugs suspected of having PDE were collected for entry into the dedicated database. Clinical analyses recommended prior to euthanasia included: complete blood counts, liver enzyme panels, and serology for Rocky Mountain spotted fever, canine ehrlichiosis, Lyme disease, canine distemper, and toxoplasmosis.

Animal Characteristics

Phenotypes were collected from owners and attending veterinarians. Incidence data was collected on 4,698 animals, together with clinical evaluations including coat color and gender. To estimate the heritability of disease, a threshold model was used to assess liability. This method assumes that a dog can be assigned to a specific disease class (unaffected/affected) when an underlying, unobservable risk (or liability) for disease exceeds a threshold of zero (0.0). For PDE, the distribution of unobservable liability was assumed to be multivariate normal in a model that included effects for sex and coat color (with classes fawn, black and unknown). Tests of the effect of gender and coat color on the liability of disease were also conducted. A mixed model Bayesian strategy outlined by Sorensen et al.14 was used to arrive at an estimate of h2 and the differences in disease liability between sex and coat color classes. Calculations were implemented through the public domain computer program MTGSAM.15 Estimation of the posterior distribution of unknown parameters employed a technique of numerical integration (Gibbs sampling).16 A total of 250,000 samples of possible heritability values were generated. The estimate was taken from the mean of every 25th iterate, after discarding the first 50,000 samples (known as the "burn-in"), for a total of 8,000 sample observations (i.e., [250,000-50,000]/25 = 8,000).

Complex Segregation Analysis

The possibility that PDE could be influenced by a segregating locus of large effect was also examined. Complex segregation analysis (Bonney17), was intended to integrate Mendelian transmission genetics at a single locus with the patterns of covariance expected in polygenic inheritance. Criteria to establish the necessary evidence for acceptance of a single locus model while reducing the risk of false positive discovery were presented by Elston et al.18 Evaluation of the models necessary for complex segregation analysis in this binary disease trait was conducted with the Bayesian software package iBay (2006, Version 1.0). The analysis was initiated with prior densities that were flat for the fixed effects of gender and coat color, and of the inverted Wishart for the unknown variances.

Results

Animals

For 4,698 dogs, clinical data, coat color, gender, and pedigrees were collected. The set included 2,875 females with 2,831 unaffected and 44 affected. There are 1,823 males with 1,809 unaffected and 14 affected. Of the Pugs with recorded coat color, 1,567 were classified "fawn" (1,512 unaffected and 55 affected) and 404 were classified "black" (401 unaffected and 3 affected). There was a lack of color records for 2,727 Pugs (2,727 unaffected and 0 affected). The median onset age of PDE is 19 months (range of 3 months to 84 months). Median survival time is 23 days with an age of death (euthanasia or natural death) ranging from 3 months to 85 months. Necropsy was performed at TAMU on 41 dogs. Remote necropsy was performed on the remaining 17 dogs; however, slides of cerebrum were forwarded to TAMU for definitive distinction between GME and PDE. Prior to death, blood work was completed including: complete blood counts, liver enzyme panels, and serology for Rocky Mountain spotted fever, canine ehrlichiosis, Lyme disease, canine distemper, and toxoplasmosis. These values were determined to be negative or within normal limits.

Estimation of Heritability

Of the total 4,698, 2,292 dogs were bred with an average inbreeding coefficient of 0.084. Results for the estimation of heritability and tests of significance for differences across sex and coat color classes (Table 1) are significant. From the posterior density of heritability, Table 1 suggests a mean density of 0.67. Moreover, 95% of samples are in the interval [0.52, 0.82], suggesting that the heritability of PDE is quite pronounced. The additional values suggest that gender and coat color are also significantly associated with disease. Additionally, the 95% HDR for the gender and coat color contrasts do not overlap zero. Positive values in Table 1 suggest that females are more likely to be affected PDE and that fawn coat colors are more frequent in affected animals than black coat colors.

Complex Segregation Analysis

A set of statistics for the complex segregation analysis of disease are presented (Table 2); there is no evidence that a major locus of large effect is segregating in this disorder. This conclusion is based on the estimated transmission probabilities for a putative major allele. Our estimates of allele transmission probabilities significantly differed from the expected Mendelian values of 1.0, 0.5 and 0.0 for transmission of the "A" allele from AA, AB and BB genotypes, respectively.18 The HDR for each of these probabilities demonstrates considerable overlap across the three putative genotypes, indicating that the transmission of this allele does not behave by the well-established rules put forth by Mendel.

Discussion

Although the clinical and pathologic aspects of PDE have been previously described, there have been no investigations into the genetic transmission or the molecular genetic mechanisms of the disease. A genetic basis for PDE has been suspected for some time, but the pathogenesis and evidence of a definitive mode of transmission remain elusive. Without basic information regarding disease transmission, breeding programs have lacked justification for the inclusion or exclusion of certain dogs. This study involved the most comprehensive set of data yet reported for PDE. Among the data collected for the 4,698 dogs are complete medical records including vaccination history, gender, color, vital statistics, and pedigree information. The requirements for voluntary inclusion consisted of necropsy with histopathology, a veterinarian's and an owner's account of disease progression, medical record submission and, when available, pedigree records. Pugs undergoing necropsy with histopathology negative for PDE were included as normal controls. Of the total dogs entered in this study, 51.1% were bred with an average inbreeding coefficient of 0.084, a value slightly higher than the predicted inbreeding coefficient for first cousin matings (0.0625), and slightly lower than the inbreeding coefficient for uncle-niece or aunt-nephew mating (0.125). This lack of random assortment in the Pug mating population expectedly yields increased homozygosity across the genome, pushing the proportion beyond that predicted by Hardy-Weinberg equilibrium. This equilibrium shift is also demonstrated in Alkaptonuria, a homozygous recessive condition. Present in the general human population at a rate of 0.002 individuals, offspring of a first cousin mating with an inbreeding coefficient of 0.0625 are expected to have an alkaptonuria frequency represented by the equation: freq (k/k) = qf + q2(1-f) in which f equals the inbreeding coefficient and q is equal to the alkaptonuria allele frequency in the population. For this example: freq (k/k) = (0.002)(0.0625) + (0.002)(0.002)(0.9375); freq (k/k) = 1.3 x 10-4

This rate is approximately 32 times greater than the random frequency of the disease, 4 x 10-6.19 Considering that the coefficient of inbreeding in Pugs studied here is higher than that of first cousin matings, it is not surprising that the heritability of PDE is high. On a scale of 0 to 1.0, 95% of the affected dogs display heritability between 0.52 and 0.82 (Table 1). In addition to demonstrating high heritability, the analysis also reveals a significant association between the dogs' gender, coat color, and heritability of PDE. The data clearly indicate that within this cohort, the majority are fawn females while the least frequent combination of affected gender and color is black males. This association initially appears unusual; however, when considering the increased popularity of fawn dogs, whose color is recessive, it may be reasonable that a spontaneous mutation has inadvertently perpetuated within the population in a recessive fashion together with color preferences.

While demonstrating that the incidence of PDE is highly heritable, the definitive mode of transmission is the parameter of primary interest. Towards this goal, disease in Pugs was examined in a Bayesian mixed-inheritance model both with and without Mendelian transmission. The analysis was performed under the pretense of PDE being caused by a single locus of major influence. The estimates of allele transmission probabilities significantly differed from the values expected under the influence of a single major allele. Specifically, HDR for each transmission probability (Table 2) demonstrates considerable overlap across the three genotypes, indicating that allele transmission does not behave in a straightforward Mendelian fashion. Recently, the lack of straightforward Mendelian inheritance has become recognized in diseases such as cystic fibrosis, and is described as complex variation.21,22 The inherent variability in disease expression can be attributed to allelic differences23,24 among affected individuals. Minor genetic differences in regulatory elements can also contribute, dramatically influencing gene expression levels.25,26,27 Considering the variable phenotypic expression of PDE, it is prudent to further analyze these possible contributions to inheritance.

Onset age of PDE ranges from 3 months through 7 years. Duration from onset of neurological signs until death can be quite variable, ranging from 24 hours to 480 days. The window of onset is wide, but expected, given a heritable condition with variable expressivity. From a genetic perspective, this demonstrates the possibility of a primary heritable factor (i.e., single locus) with modifier loci as one possibility. Numerous other alternatives to simple Mendelian inheritance also exist, a few of which are: oligogenic diseases, imprinting diseases, and mitochondrial diseases. Each category is additionally subject to genetic influences such as incomplete penetrance, phenotypic variability, locus heterogeneity, genetic modifier effects, anticipation, and emergence of phenotypes requiring environmental triggers. A common example of such variability is a disorder with high familial recurrence, holoprosencephaly. The variability ranges from prenatal lethality (36%) to obligate carriers displaying no clinical signs.28 Although many genes have been implicated, many individuals with holoprosencephaly do not have mutations within the identified causative genes.28,29,30 The absence of identifiable mutations suggests that altered regulatory elements or other genetic and environmental effects may influence the expression of holoprosencephaly.31,32,33 Environmental factors, in particular, are known to play an important role in the manifestation of phenotypes, especially in terms of exacerbating organism stress. Infection-dependent inflammatory responses may be required for numerous diseases where a genetic component has been demonstrated.20 Often believed an instigating factor in type 1 diabetes, immune responsiveness to bacterial antigens are also associated with a known genotype in some patients.36 While the specific stressor may vary, its influence on gene expression and numbers of genes may also vary.38 For analysis of PDE, strong consideration has been given to the possibility of an infectious trigger to disease instigation; however, no viral DNA has been identified in this or in previous studies.11 PCR screening for herpes-, adeno-, and parvoviruses proves negative on all included cases, arguing strongly against these viruses as triggers for PDE.

The data presented here demonstrate a strong familial inheritance for PDE. The tendency for fawn females to develop PDE more frequently than black, male Pugs is highly significant. The lack of a straightforward Mendelian form of inheritance, however, suggests the possibility of genetic modifiers or additional influences contributing to the phenotypic expression of disease. To this end, further genetic and environmental factors require analysis before a complete understanding of the genetic basis of PDE fully develops. With studies ongoing in both areas, including tests to identify susceptible genotypes and the presence of pathogenic triggers, the potential exists for the ultimate reduction, or more hopefully, the elimination of PDE.

Table 1. Marginal posterior means, modes, standard deviations and limits to the 95% highest density regions of threshold model parameters for disease in Pugs in a Bayesian analysis.

 

Heritability

Female - Male

Fawn - Black

Mean

0.67

2.52

1.49

Mode

0.62

2.33

1.42

SD

0.08

0.71

0.59

HDR 95% Low

0.52

1.22

0.40

HDR 95% High

0.82

3.97

2.66

Table 2. Marginal posterior means, modes, standard deviations and limits to the 95% highest density regions of model parameters for disease in Pugs in a Bayesian mixed-inheritance model with a completely recessive major locus, with and without Mendelian transmission of the putative major allele.

 

Polygenic
variance

Major locus
variance

Additive
effect (a)

Dominance
deviation (d)

τAA

τAB

τBB

Frequency
(q)

Mendelian Transmission

Mean

0.56

5.07

2.54

-2.54

1.0

0.5

0.0

0.85

Mode

0.11

13.92

1.72

-4.45

-

-

-

0.88

SD

0.55

2.48

0.69

0.69

-

-

-

0.05

HDR 95% Low

0.00

2.22

1.16

-5.03

-

-

-

0.68

HDR 95% High

3.14

19.02

5.38

-0.84

-

-

-

0.95

Non-Mendelian Transmission

Mean

1.19

2.86

2.50

-2.50

0.94

0.32

0.11

0.93

Mode

0.24

1.56

0.75

-2.18

0.99

0.02

0.02

0.95

SD

0.84

2.09

0.92

0.92

0.16

0.08

0.09

0.05

HDR 95% Low

0.00

0.00

0.00

-4.56

0.18

0.00

0.00

0.47

HDR 95% High

3.22

13.69

5.20

0.18

1.00

0.68

0.63

1.00

References

1.  Cordy DR, et al. Vet Pathol 1989;26:191-194.

2.  de Lahunta A. Veterinary Neuroanatomy and Clinical Neurology, 2nd ed. Philadelphia:WB Saunders Co, 1983.

3.  Kobayashi Y, et al. J Comp Path 1994;110:129-136.

4.  Beltran WA, et al. Journ Small An Prac 2000;41:161-164.

5.  Hinrichs U, et al. Tierarztl Prax 1996;24:489-492.

6.  Cordy DR. Vet Pathol 1979;16:325-333.

7.  Kornegay JN. Pug Talk 1991;6:18-22.

8.  Thomas WB. Clin Tech Sm An Prac 1998;13:167-178.

9.  Kuwabara M, et al. J Vet Med Sci 1998;60:1353-1355.

10. Summers BA, et al. Vet Neuropath 1995;95-188.

11. Schatzberg S, et al. J Vet Intern Med 2005;19:553-559.

12. Stuart S. Pug Talk 1991;1: 40-48.

13. Norton MM. Pug Talk 1992; Jan/Feb:42-43.

14. Sorensen DA, et al. Genet Sel Evol 1995;27:229-249.

15. Van Tassell CP, et al. In: U.S. Department of Agriculture, Agricultural Research Service, eds. 1995;84-86.

16. Geman S, et al. IEEE Trans Patt Anal Mach Intell 1984;6:721-741.

17. Bonney GE. Biometrics 1986;42:611-625.

18. Elston RC, et al. Ann Hum Gene 1975;39:67-87.

19. Mange EJ, et al. In: Basic Human Genetics 2nd ed. Sunderland MA: Sinauer Associates Inc., 1999:339-364.

20. Dipple KM, et al. Am J Hum Genet 2000;66:1729-1735.

21. Badano JL, et al. Nat Rev Genet 2002;3:779-789.

22. Jezequel P, et al. Mol Hum Reprod 2000;6:1063-1067.

23. Caspi M, et al. J Biol Chem 2003;278:38740-38748.

24. Cowles CR, et al. Nat Genet 2002;32:432-437.

25. Pastinen T, et al. Physiol Genomics 2004;16:184-193.

26. Ming JE, et al. Am J Hum Genet 2002;71:1017-1032.

27. Dubourg C, et al. Hum Mutat 2004;24:43-51.

28. Nanni L, et al. Hum Mol Genet 1999;8:2479-2488.

29. Edison RJ, et al. N Engl J Med 2004;350:1579-1582.

30. Shim YH, et al. Biochem Biophys Res Commun 2004;315: 219-223.

31. Roux C, et al. Am J Clin Nutr 2000;71:1270S-1279S.

32. Todd JA, et al. Hum Mol Genet 1996;5:1443-1448.

33. Reveille JD. Curr Rheumatol Rep 2004;6:117-125.

34. von Brasch L, et al. Blood Cells Mol Dis 2002;32: 309-314.

35. Schwartz PJ, et al. Circulation 2001;103: 89-95.

Speaker Information
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Kimberly Greer, PhD
Texas A&M University
College Station, TX


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