Estimated Frequency of the Canine Hyperuricosuria Mutation in Different Dog Breeds

Abstract

Background

Hyperuricosuria is a condition that predisposes dogs to urate urolithiasis. A mutation that causes canine hyperuricosuria was previously identified in 3 unrelated dog breeds. The occurrence of the mutation in additional breeds was not determined.

Hypothesis/Objectives

Identify additional breeds that have the hyperuricosuria mutation and estimate the mutant allele frequency in those breeds.

Animals

Three thousand five hundred and thirty dogs from 127 different breeds were screened for the hyperuricosuria mutation.

Methods

DNA samples were genotyped by pyrosequencing and allele-specific polymerase chain reaction methods.

Results

Mutant allele frequencies that range from 0.001 to 0.15 were identified in the American Staffordshire Terrier, Australian Shepherd, German Shepherd Dog, Giant Schnauzer, Parson (Jack) Russell Terrier, Labrador Retriever, Large Munsterlander, Pomeranian, South African Boerboel, and Weimaraner breeds.

Conclusions and Clinical Importance

The hyperuricosuria mutation has been identified in several unrelated dog breeds. The mutant allele frequencies vary among breeds and can be used to determine an appropriate breeding plan for each breed. A DNA test is available and may be used by breeders to decrease the mutant allele frequency in breeds that carry the mutation. In addition, veterinarians may use the test as a diagnostic tool to identify the cause of urate urolithiasis.

Hyperuricosuria, the excessive excretion of uric acid in the urine, is a condition that predisposes dogs to urate urolithiasis.^1–3 All Dalmatians are hyperuricosuric, and this dog breed has been extensively studied because of its predisposition for urate urolithiasis.^1–8 Hyperuricosuria is an autosomal recessive condition.^9–11 Therefore, individuals that show clinical signs of the disease must have 2 copies of the mutation. The hyperuricosuria mutation is a single base pair substitution that causes an amino acid change in the protein made by the gene SLC2A9,⁹ a known urate transporter.^12–15 Nephrolithiasis has been correlated with mutations in the SLC2A9 gene in humans^15–17 and mice,¹⁸ similar to the correlation of urolithiasis in dogs. The SLC2A9 mutation identified in Dalmatians also causes hyperuricosuria in Bulldogs and Black Russian Terrier breeds,^9,10 2 breeds that have a reported predisposition to urate urolithiasis.^6,19–21 There are no reports of recent common ancestry among the Bulldog, the Black Russian Terrier, and the Dalmatian breeds,²² indicating that this mutation may have been present in a more distant ancestor, predating the closure of stud books in these breeds. If this mutation was present before the formal closure of these breeds’ registries, it is likely present in dogs of other breeds. It is possible that cases of urate urolithiasis in these unidentified breeds may have gone undetected or have been mistaken for other types of urolithiasis.

Breed-specific prevalence of urate urolithiasis, when caused by the hyperuricosuria mutation, can be lowered over time by decreasing the frequency of the mutant allele in the breeding populations. In order to form an educated breeding strategy, the mutant allele frequency in the breed must be established. The mutant allele frequency is calculated based on the ratio between the copies of the mutation observed in the population and the total number of copies of the gene. Therefore, the mutant allele frequency can range from 0, if none of the individuals carry the mutation, to 1, if all individuals have 2 copies of the mutation. It is important to determine the breed-specific mutant allele frequency because purebred dogs are limited to breeding only within their breed and the frequency of a mutant allele in 1 breed may differ from another. For example, mutant allele frequencies for the same drug sensitivity can differ among breeds. The frequency of the MDR1 mutation causing ivermectin sensitivity ranges from 0.06 in the Old English Sheepdog to 0.55 in Collies.²³ Mutant allele frequencies are used to estimate the proportion of unaffected, carrier and affected individuals in the population by utilizing the Hardy-Weinberg equilibrium equation.²⁴

In this study, individuals from a variety of dog breeds were screened for the hyperuricosuria mutation, including the 20 most common dog breeds registered by the American Kennel Club (AKC). In the breeds in which the mutation was identified, the mutant allele frequency was estimated. Mutant allele frequencies enable breeders to decide whether they need to test their dogs and how to apply the results of DNA testing to their breeding programs. In addition to providing a tool for breeders, this test will give veterinarians a diagnostic tool for cases of urate urolithiasis and will aid in selecting appropriate treatment for urolithiasis.

Materials and Methods

Samples for this study were obtained from several sources. Initial screening for the mutation was performed using a database of DNA samples originating from blood samples collected at the University of California, Davis William R. Pritchard Veterinary Medical Teaching Hospital (UCD-VMTH). Additional samples from the 20 most common breeds registered by the AKC in 2009, as well as from breeds in which the mutation was identified, were obtained from the UC Davis Veterinary Genetics Laboratory (VGL) DNA database and from the Canine Health Information Center (CHIC) DNA repository. Samples from the VGL with a coefficient of kinship <1/8, as defined by the software Pedmine,²⁵ were selected. Because of small sample sets, all available American Staffordshire Terrier, Giant Schnauzer, Pomeranian, and South African Boerboel samples were tested, regardless of the kinship coefficient. Samples from Standard Schnauzers, Labrador Retrievers, Large Munsterlanders, Miniature Schnauzers, and Yorkshire Terriers were submitted by collaborators. The Standard Schnauzers and Labrador Retrievers are related to varying degrees (ranging from littermates to separated by > 5 generations). The remainder of the submitted samples were separated by at least 1 generation (not related as siblings, or parent and offspring).

Blood samples from dogs diagnosed with urate urolithiasis were volunteered by veterinarians and owners. These samples were not included in the mutant allele frequency estimation and were only used to verify the presence of 2 copies of the mutation in dogs with clinical disease. Animals were cared for according to the principles outlined in the NIH Guide for the Care and Use of Laboratory Animals.

DNA was extracted from cytology brushes by incubation in 200 µL of 50mM NaOH for 1 minute at 75°C, 1 minute at 80°C, 1 minute at 85°C, 1 minute at 90°C, 10 minute at 97°C, and 5 minute at 5°C. The brushes then were removed and 100 µL of 1MTris (pH = 8) was added. Blood samples collected in EDTA tubes were extracted with a commercial DNA extraction kit.^a

Samples used for the initial screening were genotyped by the pyrosequencing method as described previously by Karmi et al.¹⁰ All other samples, aside from the Large Munsterlander samples, were genotyped at the VGL with a commercially available DNA test for canine hyperuricosuria.^b The Large Munsterlander samples were genotyped by the Animal Health Trust laboratory with a commercially available canine hyperuricosuria DNA test.^c

Mutant allele frequencies were calculated based on the genotyping results from the aforementioned sample populations. Confidence intervals for the mutant allele frequencies were calculated by the software Java Applets for Power and Sample Size.²⁶ Estimation of genotypic class percentage in the population was performed on the basis of the calculated mutant allele frequencies by use of the Hardy-Weinberg equilibrium equation.

Results

A total of 3,530 dogs from 127 breeds were genotyped in this study. An initial screen for the hyperuricosuria mutation was performed in 1,486 dogs from 127 different breeds (Table 1) in order to document the presence of the mutation in additional breeds. This screen identified carriers of the mutation in the American Staffordshire Terrier, Giant Schnauzer, German Shepherd Dog, Labrador Retriever, Parson Russell Terrier, South African Boerboel, and Weimaraner breeds as well as a homozygous affected German Shepherd Dog. A more in-depth screen of 2,044 additional samples from 28 different breeds was conducted (Table 1). These breeds included the AKC’s 20 most commonly registered breeds (for 2009), breeds in which the mutation was previously identified (in the initial screen or by our collaborators) and breeds with anecdotal reports of urate urolithiasis. This screen uncovered 3 additional breeds with individuals that carry the hyperuricosuria mutation. These breeds are the Pomeranian, from the 20 AKC breeds, the Australian Shepherd, previously identified to carry the mutation by our collaborators, and the Large Munsterlander, included because an individual affected with urate urolithiasis and homozygous for the hyperuricosuria mutation was identified. The mutant allele frequency was calculated for the 10 breeds identified with the mutation and the percentage of affected and carrier individuals in each breed was estimated (Table 2). Mutation frequencies in the breeds that carry the mutation range from 0.001 in Labrador Retrievers to 0.14 in Weimaraners (Fig 1).

Table 1.

List of breeds and number of dogs tested in for the hyperuricosuria mutation.

Breed	No. Tested	Breed	No. Tested	Breed	No. Tested
Affenpinscher	5	Dandie Dinmont Terrier	9	Petit Basset Griffon Vendéen	7
Afghan Hound	3	Doberman Pinschera	109	Pit Bull Terrier	11
Airedale Terrier	7	English Pointer	1	Pomeraniana,b	88
Akita	16	English Setter	1	Poodlea	107
Alaskan Malamute	6	English Springer Spaniel	17	Portuguese Water Spaniel	2
American Eskimo Dog	1	Flat Coated Retriever	2	Puga	110
American Staffordshire Terriera,b	31	Fox Terrier	5	Red Bone Hound	4
Anatolian Shepherd Dog	3	French Bulldog	12	Rhodesian Ridgeback	9
Australian Cattle Dog	22	German Pinscher	1	Rottweilera	104
Australian Shepherda,b	142	German Shepherd Doga,b	114	Saint Bernard	8
Australian Terrier	1	German Shorthaired Pointera	96	Saluki	3
Basenji	1	Golden Retrievera	110	Samoyed	3
Basset Hound	35	Gordon Setter	1	Schipperke	18
Beaglea	65	Great Dane	9	Schnauzer	9
Belgian Malinois	1	Great Pyrenees	4	Schnauzer, Gianta,b	81
Belgian Tervuren	12	Greyhound	10	Schnauzer, Miniaturea	85
Bernese Mountain Dog	13	Havanese	1	Schnauzer, Standarda	81
Bichon Frise	8	Irish Setter	6	Scottish Deerhound	2
Bloodhound	2	Irish Terrier	1	Scottish Terrier	21
Border Collie	15	Irish Wolfhound	1	Chinese Shar-Pei	10
Borzoi	2	Italian Greyhound	11	Shetland Sheepdoga	143
Boston Terriera	46	Parson Russell Terriera,b	161	Shiba Inu	1
Bouvier des Flandres	7	Japanese Chin	10	Shih Tzua	35
Boxera	105	Keeshond	9	Siberian Husky	16
Briard	2	Kuvasz	1	Silky Terrier	2
Brittany Spaniel	7	Labrador Retrievera,b	384	Skye Terrier	10
Brussels Griffon	10	Lakeland Terrier	1	Soft Coated Wheaten Terrier	3
Bull Terrier	5	Large Munsterlandera,b	40	Staffordshire Terrier	12
Bullmastiff	3	Lhasa Apso	1	South African Boerboela,b	9
Cardigan Welsh Corgi	10	Maltesea	68	Sussex Spaniel	10
Cairn Terrier	4	Manchester Terrier	2	Tibetan Terrier	13
Catahoula Hog Dog	2	Mastiff	9	Vizsla	7
Cavalier King Charles Spaniel	5	McNab Shepherd	2	Weimaranera,b	77
Chesapeake Bay Retriever	2	Miniature Pinscher	14	Welsh Corgi, Pembroke	19
Chihuahuaa	111	Mix	73	Welsh Springer Spaniel	2
Chinese Crested	10	Newfoundland	14	Welsh Terrier	1
Chow Chow	8	Norfolk Terrier	1	West Highland White Terrier	15
Clumber Spaniel	2	Norwegian Elkhound	1	Whippet	2
Cocker Spaniel, Americana	110	Norwich Terrier	2	Wirehaired Fox Terrier	1
Cocker Spaniel, English	1	Nova Scotia Duck Tolling Retriever	3	Wirehaired Pointing Griffon	1
Collie	7	Old English Sheepdog	1	Yorkshire Terriera	90
Coon Hound	2	Papillon	9
Dachshunda	110	Pekingese	11

^aIncluded in the in-depth screen.

^bMutation was identified in the breed.

Table 2.

Observed and estimated hyperuricosuria mutation occurrence.

Breed	No. of Animals Tested	No. of Carriers Observed	No. of Affected Observed	Estimated Carriers in Population (%)	Estimated Affected in Population (%)
Labrador Retriever	384	1	0	0.26	< 0.01
Pomeranian	88	1	0	1.13	< 0.01
German Shepherd Dog	114	1	1	2.60	0.02
American Staffordshire Terrier	31	1	0	3.17	0.03
Australian Shepherd	142	5	0	3.46	0.03
Parson Russell Terrier	161	11	1	7.75	0.16
Giant Schnauzer	84	8	1	11.20	0.35
South African Boerboel	9	2	0	19.75	1.23
Large Munsterlander	40	9	1	23.72	1.89
Weimaraner	76	12	5	25.41	2.23

Fig 1.

Mutant allele frequency within breeds. Error bars indicate the 95% confidence intervals. The number of animals tested to determine the mutant allele frequency is noted in parenthesis next to the breed name.

To verify that the hyperuricosuria mutation is correlated with urate urolithiasis in the breeds identified with the mutation, samples from 3 Parson Russell Terriers, 1 Weimaraner, and 1 Large Munsterlander that were diagnosed with urate urolithiasis were genotyped and all 5 dogs were found to have 2 copies of the hyperuricosuria mutation.

Discussion

The hyperuricosuria mutation had been identified previously in 3 unrelated breeds: the Dalmatian, Bulldog, and Black Russian Terrier.^9,10 In order to test our hypothesis that the mutation is present in additional breeds, we initially screened 1,486 dogs from 127 different breeds. A more in-depth screen of 2,044 additional samples was later performed to determine both the mutant allele frequency in breeds identified with the hyperuricosuria mutation, and the presence of the mutation in the 20 most common breeds. We identified a total of 10 additional breeds in which the hyperuricosuria mutation was present: American Staffordshire Terrier, Australian Shepherd, Giant Schnauzer, German Shepherd Dog, Labrador Retriever, Large Munsterlander, Parson (Jack) Russell Terrier, Pomeranian, South African Boerboel, and Weimaraner.

The hyperuricosuria mutation causes an inborn error of metabolism that increases the amount of uric acid in the blood and urine of affected dogs and predisposes them to urate urolithiasis.⁹ Uroliths can be composed of different mineral types. The composition of the urolith can be determined by quantitative crystallographic analysis,²¹ which allows appropriate treatment.²⁷ None of the breeds identified in this study have been reported to have a predisposition to urate urolithiasis.^6,20,21 However, a study in Weimaraners reported unexplained massive urate crystalluria in an immunodeficient male puppy.²⁸ Although urate urolithiasis was not reported and the dog was not tested for hyperuricosuria, it is likely that hyperuricosuria was the predisposing factor for the urate crystalluria, because uric acid is not normally found in high amounts in the urine of dogs. Determination of the predisposition of a breed to a specific urolith type is based on the incidence of the urolith in that breed as compared with other types of uroliths, and compared with other dog breeds.^1,20,21 In the breeds identified in this study, urate urolithiasis might have gone unrecognized if uroliths were not submitted for quantitative crystallographic analysis. Alternatively, background genetic variation might influence some breeds affected with hyperuricosuria to be at a lower risk for developing clinical signs for urate urolithiasis, compared with other breeds. In addition, for the breeds that have a low mutant allele frequency, such as Labrador Retrievers, the percentage of the population affected with hyperuricosuria is <0.01%. The portion of the affected individuals that would show clinical signs for urate urolithiasis is likely to be even less, based on studies in Dalmatians. Within the Dalmatian breed, males are 16.4 times more likely than females to be affected with urate uroliths¹ and <35% of all males will show clinical signs for urolithiasis.^1,5 Therefore, a predisposition for urate urolithiasis in breeds with a low mutant allele frequency may never be identified.

The accuracy of the calculated mutant allele frequency for each breed is represented by the 95% confidence interval. The range of the 95% confidence interval is dependent on the sample size tested and how common the mutation is in the breed. For example, for the South African Boerboel breed, only 9 samples were available for testing and the mutation was carried by 2 individuals. Therefore, whereas the mutant allele frequency is 0.11, the 95% interval is between 0 and 0.33. In contrast, in the Weimaraner breed, 76 individuals were tested and 17 were found to have the mutation. The calculated mutant allele frequency in Weimaraners is 0.15 and the 95% confidence interval is between 0.07 and 0.23. As a rule, the higher the mutant allele frequency, the higher the chance of individuals being identified with clinical signs of urate urolithiasis. Although none of the breeds identified in this study have been reported to have a predisposition to urate urolithiasis,^1,20,21 the mutant allele frequency in Weimaraners (0.15) and Large Munsterlanders (0.14) is close to the mutant allele frequency in Bulldogs (0.16),¹⁰ a breed well documented for its predisposition to urate urolithiasis.^1,20,21 Dogs affected with urate urolithiasis were identified from the Weimaraner and Large Munsterlander breeds as well as from the Parson Russell Terrier breed during the course of this study. These samples were tested and determined to be homozygous for the hyperuricosuria mutation, providing further evidence that the hyperuricosuria mutation is correlated with urate urolithiasis in these breeds.

The frequencies of the hyperuricosuria mutation found in the different dog populations sampled in this study are an estimation of the mutant allele frequencies in these breeds. It is possible that there was an ascertainment bias in sample collection that would affect the estimated mutant allele frequency, based on the source from which the samples were obtained. Samples originating from the VGL may be biased geographically, because some of the samples were obtained by VGL personnel at local dog shows. However, samples at the VGL also are mailed in from across the United States, decreasing the geographical bias that may otherwise be present. Although samples obtained from the CHIC DNA repository may be biased toward dogs that are bred for the show ring, requests for samples for this database are made through the Orthopedic Foundation for Animals website as well as through the parent clubs of the breeds. Access to these calls for samples is available to all dog owners, regardless of their dog show or trial area of interest (eg, conformation, agility, hunting) and whether or not they plan to breed their dogs, increasing the likelihood that any dog owner interested in advancing canine health will contribute samples. Although some bias in the samples included in this study may be present, by utilizing more than 1 source for samples as well as using databases that allow and encourage submission from the general public, the inaccuracy of the allele frequency estimate was minimized. In addition, by providing 95% confidence intervals, the predicted accuracy of the estimated mutant allele frequency is presented.

The hyperuricosuria mutation was identified in more than 1 individual in all breeds identified with the mutation except for the Pomeranian, American Staffordshire Terrier, and Labrador Retriever, where only 1 carrier dog was identified. Identification of a single sample in the breed is indicative of the presence of the mutation in the breed, as long as the sample is from a purebred dog of that breed. In Pomeranians, the dog carrying the mutation is a purebred Pomeranian, and it is probably the only dog identified with the mutation because of the low mutant allele frequency in the breed. The American Staffordshire Terrier sample is from a dog seen by the reproductive service at the hospital and thus is likely to be from a purebred dog. In this breed, it is probable that only a single individual was identified because a relatively low number of samples were genotyped. The origin of the carrier Labrador Retriever sample is from the UCD-VMTH database, which reports the breed identity based on owner declaration. Because there was no indication in this patient’s medical record that it is a purebred Labrador Retriever, it is possible that this is a mixed-breed dog. Because of this uncertainty, 268 samples from verified purebred Labrador Retrievers were tested but the mutation was not identified in this sample set. Therefore, the hyperuricosuria mutation in Labrador Retrievers is either in very low frequency or not present at all. The presence of the mutation in all 3 breeds could be verified if additional individuals carrying the mutation are identified.

Although the mutation was not identified in some of the breeds included in this study, we cannot conclude that the mutation is not present in those populations. The mutant allele frequency may be very low in these breeds and thus undetectable with the sample size tested. In these cases, the mutant allele frequency is smaller than

$${(2\times \text{number of dogs genotyped})}^{-1}$$

For example, 143 Shetland Sheepdogs were genotyped and so the mutant allele frequency in this breed is <0.003. This study focused on 28 breeds and it is probable that the mutation is present in additional breeds that were not included. Therefore, any dog affected with urate urolithiasis may have 2 copies of the hyperuricosuria mutation and, after verification by DNA testing, can be treated according to the same protocols established for Dalmatians.^3,6

In breeds identified with the mutation, selection against hyperuricosuria is possible by DNA-based testing and has the potential to decrease the production of affected individuals and the frequency of urate urolithiasis in these breeds. A genetic test for canine hyperuricosuria is commercially available and may be used by breeders to select animals for breeding. Selection against the mutation should be done with consideration for the mutant allele frequency and the breed’s population size. In breeds with a high mutant allele frequency, such as Weimaraners, a gradual selection is recommended in order to prevent a strong decrease in the overall genetic diversity of the breed. Because hyperuricosuria is a recessive trait, gradual selection can be achieved by breeding with carriers, provided they are mated to DNA-tested, clear dogs. In breeds with a very low mutant allele frequency and a large population, such as German Shepherd Dogs, selection can be done more rapidly, by breeding only dogs clear of the mutation, without compromising the desired genetic diversity of the breed. Veterinarians also may use the DNA test in order to investigate the cause of urate urolithiasis in affected dogs. The hyperuricosuria mutation is not the only cause of urate urolithiasis, and conditions such as portosystemic shunts and hepatic microvascular dysplasia also may predispose dogs to this disease.⁶ The DNA-based test may be used as a noninvasive and relatively inexpensive tool to differentiate between such cases.

Abbreviations

AKC

American Kennel Club

UCD-VMTH

University of California, Davis William R. Pritchard Veterinary Medical Teaching Hospital

VGL

Veterinary Genetics Laboratory