The Genetic & Molecular Basis of Urate Calculi Formation in Dalmatians
ACVIM 2008
Danika Bannasch, DVM, PhD
Davis, CA, USA

The Dalmatian is unique among dogs since it cannot convert uric acid to allantoin during the degradation of purines. This leads to hyperuricosuria(huu) and relative hyperuricemia as compared to other dogs. This phenotype is seen in all members of the Dalmatian breed.1-3 All purebred Dalmatians tested excrete 20 fold higher amounts of urate, the salt of uric acid, in their urine than normal dogs. It is one of the oldest known inherited traits in dogs, having been first described by S.R. Benedict in 1916.4 While studying the metabolism of uric acid, Benedict discovered that acidifying the pH of Dalmatian urine resulted in the formation of uric acid crystals. Benedict was interested in the Dalmatian dog as a potential animal model for the study of uric acid metabolism and excretion in humans. Dalmatian purine metabolism has been extensively studied over the years; however only recently has the molecular nature of the disease been determined.

Purine Metabolism

Purines are important building blocks for cells. They form the basis of nucleotides that are used to make RNA and DNA as well as energy sources such as ATP, GTP, cAMP, and NADH. Purines are provided by an organism's diet and can also be salvaged and synthesized from the breakdown of other purines (AMP and GMP). In dogs, excess purines are catabolized and excreted in the urine in the form of allantoin. In Dalmatians, humans and great apes, the final product of purine catabolism is uric acid. In humans and great apes the cause of this change in catabolism is due to the absence of the enzyme urate oxidase which catalyzes the final step in this pathway (Figure 1). Uric acid freely circulates in the form of urate, the salt of uric acid, in the plasma where it serves as a free radical scavenger. Although uric acid has evolved in humans to be the main breakdown product of purines, there appear to be negative effects of this change in purine catabolism. High levels of urate predispose humans to gout and nephrolithiasis. In addition, uric acid levels have been correlated with hypertension, vascular disease and the metabolic syndrome.5 Serum uric acid levels are higher in Dalmatians than in other dogs but are not as high as they are in humans. In Dalmatian dogs, the negative effects of this change in purine catabolism appear to be limited to the formation of urinary calculi.

Figure 1.
Figure 1.

Biochemical steps in the degradation of purines (enzymes that catalyze the reactions are boxed).
 

Transport

The level of urate in the blood is controlled by differences in the production of urate (influenced by diet) as well as its excretion in the urine. In humans and great apes the enzyme that converts uric acid into allantoin is absent, resulting in elevated levels of uric acid. In the kidney, urate is filtered by the glomerulus and then reabsorbed in the proximal tubules where it re-enters circulation. There are species-specific differences in relative amounts of reabsorption and secretion in the proximal tubules which makes using animal models in this area of research challenging. The proteins involved in the reabsorption of urate from the ultrafiltrate are just beginning to be discovered. Urate must be transported across the apical membrane of the proximal tubules and then across the basolateral membrane in order to re-enter circulation. Plasma urate levels may be controlled at the level of the proximal tubules of the kidney; therefore a complete understanding of urate transport is essential to understanding the regulation of blood urate levels.

In addition to the production and excretion of urate, there are species-specific differences in the handling of urate by the kidney. Urate is filtered by the glomerulus resulting in high levels of urate in the glomerular filtrate. Dogs and humans both undergo bidirectional transport of urate along the nephron resulting in net reabsorption of urate from the glomerular filtrate. Other species (pigs and rabbits) undergo net secretion of urate. Blood levels of uric acid can be altered by changing the amount of urate reabsorbed by the proximal tubules.

Since the Dalmatian phenotype involves liver metabolism and urinary excretion of uric acid, researchers studied liver and kidney tissues to determine the organ responsible for the problem. By transplanting liver or hepatocytes between Dalmatian and non-Dalmatian dogs, it is possible to correct the defect in Dalmatians and induce hyperuricosuria in wild-type dogs.6 When similar studies were done with kidney transplants, the affected phenotype could be altered but not corrected completely.7 Giesecke & Tiemeyer studied the biochemical function of urate oxidase (uox), the enzyme that converts uric acid into allantoin. These researchers demonstrated that urate oxidase functions in Dalmatian liver homogenates but not in liver slices. Consequently, it was suggested that the Dalmatian phenotype could be explained by the lack of function of a urate transporter. If a urate transporter is not functioning in Dalmatian liver, uric acid fails to enter the appropriate cells for degradation.8-10 Free-flow micropuncture experiments were used to demonstrate that there is a deficiency of proximal tubular reabsorption of urate in Dalmatian kidneys.2 In fact, prior to the organ transplantation experiments the kidney was believed to be the primary site of action of Dalmatian huu. Ultimately, it appears that Dalmatians lack the ability to transport urate into both the liver and kidney proximal tubules. Dalmatian dog erythrocytes have been shown to transport urate normally; therefore Dalmatians do not have a generalized defect in urate transport.10

Clinical Disease

The clinical consequence of hyperuricosuria is that Dalmatians are predisposed to forming urinary stones composed of urate, which are salts of uric acid. The relative hyperuricemia in the Dalmatian does not cause medical problems the way that it does in people, probably due to the lower serum concentration of uric acid in the Dalmatian. The stones can be life threatening if they block the urethra of male Dalmatians. Surgical removal of the stones is momentarily curative but long-term medical therapy is often necessary. Medical management can consist of diet change (lower purine content), urine acidification, lowering urine specific gravity, and/or treatment with drugs that decrease uric acid production. One such drug is allopurinol, an inhibiter of xanthine oxidase, which works by decreasing uric acid production while increasing the excretion of xanthine and hypoxanthine (Figure 1). The dose of allopurinol must be carefully regulated during the dog's lifetime since a dose that is too high leads to accumulation of xanthine, which can also form insoluble stones, in the urine. Monitoring uric acid production in a quantitative fashion is done by 24 hour urine collection and subsequent measuring of uric acid levels.

Although all Dalmatians have the mutation for huu, not all Dalmatians have clinical signs of urinary disease. The majority of female Dalmatians never show clinical signs of urinary disease. In a survey done of our hospital population, 26.5% (78/294) of male Dalmatians were seen for urinary stone disease compared to no affected female Dalmatians.11 This is most likely due to anatomical differences between the sexes and the relatively small size of urate calculi.

Mode of Inheritance

The mode of inheritance of hyperuricosuria was analyzed by crossing Dalmatians to other breeds of dog.12 The puppies produced in these crosses had normal urate production in their urine consistent with a simple autosomal recessive mode of inheritance. When these hybrid dogs were crossed back to Dalmatians the numbers and genders of normal and high urate producers were equal, indicating the disorder was in fact inherited as a simple autosomal recessive. Hyperuricosuria is a unique inherited disease among dogs since all members of the Dalmatian breed are affected. This is a very unusual situation in a purebred dog breed. Most inherited diseases seen in purebred dogs are inherited as simple autosomal recessive disorders that segregate within a breed. One theory that has been proposed for this phenomenon is that the gene for hyperuricosuria is linked to a gene involved in the striking spotting pattern seen in the Dalmatian. Consequently, unbeknownst to the early breeders of Dalmatians, they were unwittingly selecting for hyperuricosuria as they selected for spotting.

Interbreed Cross

All members of the Dalmatian breed are homozygous for huu, making it difficult to study genetically since it is not segregating within the breed. Crosses were performed by a scientist and dog breeder, R. S. Schaible, to introduce the normal gene into the Dalmatian breed. It was postulated that the trait was fixed in the breed due to selection for larger spot size. This cross was started in 1973 when a female Dalmatian was crossed to a male Pointer. The F1 progeny were tested by measuring the urinary uric acid levels at six weeks of age3 and all had the wild-type phenotype (low levels of uric acid). A single F1 was then backcrossed to a Dalmatian and offspring with the wild-type phenotype and that conformed to the Dalmatian breed standard were selected to backcross to purebred Dalmatians. This breeding program has been ongoing for 12 generations, producing backcross dogs that exhibit the breed characteristics of Dalmatians but carry the wild type allele for huu.3 The spotting pattern does appear to be different between high and low uric acid dogs in the backcross, suggesting that the trait was fixed in the breed due to selection for larger spot size. The backcross dogs segregate a single copy of the wild-type allele from the Pointer together with the affected Dalmatian allele, providing a powerful genetic tool for linkage analysis.

Mapping the Disease

A putative urate transporter gene, LGALS9, has been excluded from huu by segregation analysis using the Dalmatian X Pointer Backcross dogs.13 Given the exclusion of the putative urate transporter gene as a candidate for huu in Dalmatians, urate oxidase (uox) was pursued as an alternative candidate gene. Urate oxidase appeared to be an appealing candidate gene because it encodes the enzyme that converts uric acid into allantoin in all mammals. Although Giesecke & Tiemeyer9 demonstrated that urate oxidase functions in Dalmatian liver homogenates, there was no function in liver slices, implying that there may be an alteration in function between Dalmatian and non-Dalmatian urate oxidase. A uox (-/-) mouse model shows hyperuricosuria and hyperuricemia similar to the phenotype seen only in the Dalmatian breed and in humans.14 This knockout phenotype supports the hypothesis that the silencing of uox in different species results in a comparable phenotype to Dalmatian huu. The exclusion of uox as a candidate gene for Dalmatian huu was accomplished using DNA samples from the backcross dogs for segregation analysis.15 In total, six markers surrounding the uox gene were genotyped on 25 progeny from the backcross family. There was no association between the markers' haplotype and the huu phenotype. In order to statistically analyze the genotyping results, Multipoint LOD score analysis was performed using the Mendel software. Each multipoint interval tested resulted in a LOD score of less than -2, with the most significant being -35.49. The uox gene has been excluded as a candidate for hyperuricosuria in Dalmatians based on negative LOD scores, lack of segregation in the backcross family, and the absence of changes within the coding sequence of affected and normal dogs.

The Pointer x Dalmatian backcross dogs provide a unique genetic tool to evaluate candidate genes for the hyperuricosuria defect since the animals in the project segregate the defective Dalmatian allele. In order to determine the chromosomal location of the huu locus, we performed a genome scan with microsatellite markers multiplexed by researchers at the Veterinary Genetics Laboratory (UC Davis) on the backcross animals. In total, 148 markers were genotyped in this family. Two point LOD scores were estimated for these markers and only one was significantly linked to the phenotype. This marker is located on CFA 03. We have confirmed the linkage with additional markers in the area that are polymorphic and have verified that this is the correct chromosome location of the hyperuricosuria locus. Even without taking into account the 5-11 generations of backcrossing, the LOD score at theta=0 is 6.55 for two different markers and the phenotype.16

Fine structure mapping on CFA 03 has been performed using additional backcross dogs narrowing the interval to a small (2.4 Mb) region containing 19 predicted genes. Since Dalmatians are fixed (homozygous) for huu, it was expected that an area of homozygosity (in which both alleles are identical) around huu would be identified. Blocks of linkage disequilibrium in purebred dogs extend between several megabases in rare breeds that have small population sizes to several kilobases in popular breeds.17 Given that Dalmatians have a moderate population size, we hypothesized that the homozygous region surrounding huu would be smaller than 2.4 Mb and that it could therefore shorten the list of potential candidate genes. This approach has been successful and the gene and mutation that cause Dalmatian hyperuricosuria have been identified. Additional breeds know to form urate calculi have also been shown to have the same mutation as the Dalmatian dogs, providing opportunities for breeders of non-Dalmatian breeds to eliminate this disease.

Within the Dalmatian breed all dogs are homozygous for the huu mutation. The only way to eliminate or reduce the prevalence of the disease within the Dalmatian bred is to allow registration of the Dalmatian x Pointer backcross dogs which don't carry the mutation but have the outward appearance of purebred Dalmatians.

References

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Speaker Information
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Danika Bannasch, DVM, PhD
University of California
Davis, CA


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