The Dalmatian Backcross Project- Brief Summary of Research (November, 2005)
Background
Dalmatians have a unique urinary metabolism defect. Most dogs produce allantoin
in their urine from the breakdown of proteins that they eat. Dalmatians produce
a compound called uric acid in their urine from the breakdown of proteins.
The only other mammals that produce uric acid are humans, great apes and
some new world monkeys. The elevated level of uric acid in the urine is termed
hyperuricosuria. Both male and female Dalmatians can form bladder stones
composed of urate, the salt of uric acid. The stones can be life threatening
in male Dalmatians if they cause a blockage of the urethra preventing the
dog from urinating. Female Dalmatians rarely develop clinical signs associated
with hyperuricosuria. The treatment for Dalmatians that form stones requires
lifelong medication and frequent visits to the veterinarian for urine testing
(Sorenson and Ling, 1993). The goal of this project is to identify the genetic
cause of the urinary metabolism defect in the Dalmatian.
Hyperuricosuria was first identified in 1916 making it one of the oldest
recognized inherited diseases in dogs (Benedict, 1916). All Dalmatians that
have been tested have hyperuricosuria and are postulated to be homozygous
for the recessive mutation that causes this disease. Hyperuricosuria (huu)
in Dalmatians is inherited as an autosomal recessive trait. The mode of inheritance
was established when Dalmatians were crossed to other breeds and F1 dogs
showed normal levels of uric acid. When these hybrid dogs were crossed back
to Dalmatians, the numbers of normal and high level uric acid phenotypes
and the gender distribution of the phenotypes were equal (Onslow 1923, Keeler
1940, Schaible 1986).
In order to identify the location of the gene responsible for hyperuricosuria
it is necessary to have animals that segregate the disease. Since all Dalmatians
are fixed for the trait the only way to accomplish this is through a cross
to a dog that is normal for the trait. R. S. Schaible has performed just
such a cross in order to correct the Dalmatian disease. He crossed a single
pointer male to a
Dalmatian in 1973. Offspring from this cross were backcrossed to a different Dalmatian. In the next generation, the puppies were evaluated for urate production by testing the urine and measuring the uric acid concentration as well as creatinine concentration (see progeny testing). Puppies that were the most like the Dalmatian standard and that were normal uric acid producers were used in subsequent generations for breeding to Dalmatians. This backcross has been carried out for 11 generations with selection for normal uric acid production in each generation. The backcross dogs provide a powerful genetic tool not usually available in dog genetics to identify the disease causing gene.
Progeny testing
Puppies from each generation of the backcross have their urine tested in duplicate at 6 weeks of age. Uric acid levels as well as creatinine levels are measured and the ratio of uric/creatinine is used to assess the puppy’s phenotype at the hyperuricosuria locus (huu). This method would not work well in adult dogs (Ling, unpublished results) however in young puppies there appears to be good separation between normal values and hyperuricosuric values. Duplicate samples are used to evaluate the high uric acid producers since their levels can vary greatly with metabolism. The low uric acid producers are always low and have very little variance. The following graph shows the average of two uric acid/creatinine ratios of different urine samples for 14 low backcross puppies and 14 high backcross puppies. There is a highly significant difference between low uric acid backcross Dalmatians and high backcross Dalmatians (p<0.0001).
Molecular genetics
Although the huu phenotype involves hepatic
degradation of purines and renal excretion of uric acid, transplantation
studies localized Dalmatian huu to the liver. Transplanting
livers or hepatocytes between Dalmatians and non-Dalmatian
dogs corrected the defect in Dalmatians and induced hyperuricosuria
in wild-type dogs (Kuster 1972). When similar studies were
done with kidney transplants, they did not alter the affected
phenotype (Appelman 1966).
The liver enzyme that converts uric acid into allantoin, urate
oxidase (uox), was found to be functional in Dalmatian liver
homogenates but not in liver slices (Giesecke & Tiemeyer 1984). This finding
suggested that perhaps a defective urate transporter, which resulted
in the failure of uric acid to enter the appropriate cells for degradation,
was the cause of the Dalmatian phenotype (Vinay et al 1983, Giesecke & tiemeyer
1984, Kocken et al 1996). One putative urate transporter gene,
galectin 9, was excluded from huu by linkage analysis using DNA
from the Dalmatian x Pointer backcross dogs (Bannasch et al 2004).
Following the exclusion of galectin 9, the backcross pedigree
was used to evaluate, and subsequently exclude, the uox gene
as a candidate for Dalmatian huu (Safra et al 2005). Uox was
an appealing candidate gene, given that during primate evolution
it was silenced, resulting in high levels of uric acid in those
species (Oda et al 2002).
Genome scan and linkage analyses were performed in order
to map hyperuricosuria in the Dalmatian dog. A total of 25
backcross dogs, for which family data, phenotype and DNA
samples were available, were genotyped with 215 markers.
PCR products were obtained for 148 markers which were used
for subsequent analysis, and parentage was verified for each
marker. The genome scan resulted in a significant linkage
with a LOD score of 3.99 for a particular marker. Additional
markers located up to 20.73 Mb proximal and 9.16 Mb distal
to the original linked marker were genotyped on a total of
36 family members (as 11 additional DNA samples became available).
A maximum LOD score of 6.55 was obtained when the 11 additional
samples were genotyped (Safra, Schaible and Bannasch, manuscript
under review).
Fine structure mapping of the region around the linked marker
was performed by haplotype analysis of 36 backcross dogs. Two
recombinant individuals distinguish the region proximally and
distally to huu. These haplotypes enabled us to define an interval
of 3.3 Mb, flanked by single recombination events, that contains
the hyperuricosuria locus. At present, there are 24 predicted
genes located within this region.
Genetic testing of backcross progeny
We continue to collect DNA samples as well
as urine from backcross puppies in order to help us identify
recombination breakpoints that will further localize the huu
locus. At present, dewclaws are collected and DNA is extracted
from them. Because we have a set of markers surrounding the huu
locus we can effectively determine the phenotype (low or high
uric acid) of the puppies using molecular markers by 1 week of
age. Recombinant animals which are very rare are also identified.
These puppies’ urine test results are essential for our work
and are the only means to determine the dog’s uric acid level.
Eventually, when the mutation is identified, only molecular testing
will be necessary to determine the dog’s genotype. We have
never identified a discrepancy between a puppy’s urine
tested phenotype and its genotype (molecular test).