**4. Methods to identify the PDP1 mutation in Clumber spaniels**

## **4.1 Development of PDP1 assay in human tissue**

Pyruvate dehydrogenase complex (PDH) plays a pivotal role in metabolism and is highly regulated by the concentration of its substrates and products. It is also

### *Founder Effect: Breeding a Dog for the Elderly Gentleman Reveals an Animal Model… DOI: http://dx.doi.org/10.5772/intechopen.113912*

regulated by phosphorylation and dephosphorylation events, by kinases and phosphatases, respectively. In 1975, Robinson and Sherwood performed biochemical assays for PDH activity in tissue samples isolated from a young patient with metabolic acidosis as a result of elevated blood lactate and pyruvate [22]. In summary, the assay for PDH activity was performed in three parts. The first assesses the activity of the native PDH complex in tissue homogenate. Next, the homogenate is incubated with ATP before assaying PDH activity, to activate kinase phosphorylation which inhibits the PDH complex. Lastly, the homogenate is incubated with calcium before assaying PDH activity, to activate the phosphatase which switches on PDH activity. Their results showed that the patient's PDH complex could not be activated by calcium and proposed that the deficit was a result of defective pyruvate dehydrogenase phosphatase deficiency. After that publication, many physicians around the world sent tissue samples to the Robinson laboratory for the diagnosis of patients with congenital lactic acidosis. In 2005, the fibroblasts of two brothers with congenital lactic acidosis were sent to the Robinson lab for testing, as reported by us [3]. In summary, native PDH activity was 25% of control which could not be restored by preincubation with calcium. Maj and coworkers of the Robinson lab generated recombinant pyruvate dehydrogenase phosphatase isoform 1 (PDP1) and isoform 2 (PDP2) proteins. Patient PDH activity was restored to normal after incubation with recombinant PDP1, suggesting a defect not in the PDH complex itself but in the regulatory PDP1 protein. Mutational analysis was performed and a homozygous, pathogenic mutation in *PDP1* was identified. Recombinant PDP1 protein was also used to generate a polyclonal antibody. This was used for Western blot analysis and subsequently showed a large reduction in PDP1 protein in patient cell lysates [3].

## **4.2 Development of PDP1 assay in canine tissue**

Two years after the first genetic cause of PDP1 deficiency in humans was identified, Shelton sent tissue samples from four related Clumber Spaniels to the Robinson lab for investigation. The dogs suffered from profound exercise intolerance with elevations in lactate and pyruvate causing metabolic acidosis. Cameron and Robinson's lab coworkers performed PDH activity assays and restored PDH activity after incubation of cell lysates with recombinant PDP1 protein, as was performed for human tissue samples. Western blot analysis showed a complete loss of PDP1 expression. The affected dogs were found to be homozygous for a mutation in the *PDP1* gene. The mutation c.754C > T created a premature stop codon p.Q252X was identified by gene sequencing. The mutation in the *PDP1* gene destroyed a restriction site, and a genotypic test was created, all methodology as previously reported by us [4]. The test is known as RFLP or restriction fragment length polymorphism. The generation of this test allowed for the ability to genotype the Clumber spaniel for this mutation. A pedigree analysis for five generations is shown (**Figure 2**). A full pedigree of all dogs tested in the Robinson lab is shown for the first time (**Figure 3**). Affected dogs typically present between 15 weeks to 1 year old with exercise intolerance, described as the unwillingness to exercise or play after 5 minutes. Activity may resume after rest, but the dog will tire again quickly. Neonatal deaths have been reported in the littermates of affected dogs. Affected dogs are recommended to be fed a low-carbohydrate, high-fat diet. Carriers of the mutation do not show any clinical features [4].

#### **Figure 2.**

*Pedigree of five generations of clumber spaniels and genotyping for the c.754C > T PDP1 founder mutation. Red symbols represent individual dogs that were genotyped. Individuals of unknown sex are represented by diamonds. Carriers are identified with small circles within the symbol and homozygous affected individuals are represented by solid red symbols. The individual with red diagonal strips displayed clinical features but could not be tested. Separate breeding with the same parents is identified by small Roman numerals. This figure was originally published by us in molecular genetics and metabolism, vol 90, Cameron et al, identification of a canine model of pyruvate dehydrogenase phosphatase 1 deficiency, pages 15–23, copyright Elsevier, (2007) [4].*

#### **Figure 3.**

*Popular sire effect was seen in a small population of Clumber spaniels and genotyping for the c.754C > T PDP1 founder mutation. Red symbols represent individual dogs that were genotyped. Individuals of unknown sex are represented by diamonds. Carriers are identified with small circles within the symbol and homozygous affected individuals are represented by solid red symbols. The individual with red diagonal stripes displayed clinical features but could not be tested. The particular sire highlighted in gold is an example of a 'popular sire' and was mated 11 times within this pedigree. This data was collected during and after the development of the RFLP genetic test for the heritable PDP1 mutation in the Clumber spaniels [4]. All data have been de-identified to preserve the confidentiality of the animals and their owners.*

*Founder Effect: Breeding a Dog for the Elderly Gentleman Reveals an Animal Model… DOI: http://dx.doi.org/10.5772/intechopen.113912*
