Founder Effect: Breeding a Dog for the Elderly Gentleman Reveals an Animal Model of a Human Genetic Disorder

Abstract

Animal models of genetic disorders that have risen due to selective breeding can be used as a valuable model to teach the basic concepts of population genetics. The Clumber Spaniel is a breed of dog created in the mid-1700s by the 4th Duc du Noailles. He selectively bred this dog for the elderly gentleman. This sleepy-looking breed survives today, though 1% suffer from severe exercise intolerance due to an autosomal-recessive founder mutation in the pyruvate dehydrogenase phosphatase 1 (PDP1) gene. PDP1 deficiency was long suspected to be a human metabolic disorder and described at the molecular level in 2005 by Robinson and coworkers. The Robinson group later identified a founder mutation within the PDP1 gene of the Clumber spaniel. This case clearly illustrates how a detrimental mutant allele in a small population, when selecting for phenotype, can persist in the progeny of that group. In this review, we discuss the origin of the “Founder Effect” theory and present an example of how a bottleneck that occurred during the selective breeding of the Clumber spaniel over 250 years ago led to the current genetic status of the breed. Today, genotyping can help reduce the incidence of PDP1 in the Clumber breed.

1. Introduction

Healthy genetic diversity of populations, in humans and all other animals, is subject to fluctuations in population size. In a large population, random mating will occur, and allelic changes that are not compatible with life, or of low fitness, will be removed from the gene pool. If a population becomes somehow limited by the environment, natural selection may influence allelic changes to either increase or disappear. Charles Darwin brought his idea of natural selection forth with the publication of his book “On the Origin of Species”. Here he defined that natural selection is a process whereby the population of an organism will adapt and change to suit an environment and that offspring tend to resemble their parents [1]. Notably, mutation is the source of allelic variability and beneficial mutations increase fitness, whereas deleterious mutations reduce fitness. A limited population, or founder population due to some type of bottleneck or separation from the larger parent population, may suffer a loss of genetic variation and thus be closed off to genetic diversity. In a small and limited population, deleterious mutations may accumulate. The Wright-Fisher Model, named for the early pioneers of theoretical population genetics, describes allelic frequencies of a single gene assuming random mating, nonoverlapping generations, and no selection or mutation. This model also considers variations in population sizes and how a sudden reduction in the size may distort allelic frequency. The distortion may lead to allelic variants which were found in extremely low frequency within the general population yet relatively common in isolated groups. In a population with a finite number, a deleterious allele can fix, and thus reduce its fitness. In the absence of outcrossing, the fitness of the group may continue to reduce, up to and including extinction, under the load of the deleterious alleles. There are many examples of inherited human diseases where a pathogenic variant is found at a higher frequency in particular groups or in populations that have an elevated level of consanguinity.

In animals, a phenomenon called the “popular sire” describes the reproductive dominance of specific animals, increasing the likelihood of later matings between related animals. Applying selective pressure based on the phenotypic attributes of the popular sire within a small population of animals markedly restricts genetic diversity. This becomes problematic when there is an increase in the incidence of harmless recessive alleles, or carriers, leading to an increased potential for progeny to inherit the recessive allele in a homozygous fashion, thus resulting in an autosomal recessive disorder. Corrective measures are currently being put in place by kennel clubs around the world such as pedigree analysis, coefficient of inbreeding calculations, and the development of single nucleotide polymorphism (SNP) arrays are currently being used to manage the diversity of breeds and control heritable disorders. Autosomal recessive disorders, which are known to a specific breed can be tested to identify carriers and reduce the numbers of affected pups born. The Clumber spaniel is one of the oldest of all British spaniel breeds, with over a 250-year-old history of joining the hunt with many of Europe’s crowned heads and aristocracy beginning in the Georgian era. It is not a common breed, but better known to sporting gundog enthusiasts, being bred originally for work during the hunt rather than for appearance at show. The breed was named after Clumber Park, originally part of the Sherwood Forest and home to the Dukes of Newcastle in Nottinghamshire, England. First arriving in England between 1763 and 1768, the dog was selectively bred in the kennel of the 2nd Duke of Newcastle to work the undergrowth of the Sherwood Forest during the hunt, to be strong, slow, and steady during the gunfire, and have an exceptional nose for the game [2].

When a large population is suddenly reduced, for a variety of reasons, the resulting population will experience a reduction in the genetic variability known as Bottleneck and Founder Effects. Selective crossbreeding of purebred animals will increase the frequency of rare mutations, or alleles, which may cause diseases similar to the Founder Effect. Events that cause a group of humans to become isolated from a larger population, in time, will similarly increase the frequency of rare mutations. For canines and humans alike, autosomal recessive disorders require progeny to inherit two mutated alleles before the disease is expressed (homozygosity). Here, we review the bottleneck events that took place during the development of the Clumber spaniel breed. We describe how selective breeding for traits important for gun hunting, such as slow and steady with a specific nose, has led to the increased frequency of mutations leading to extreme exercise intolerance in the homozygous state. The particular mutation of the autosomal recessive disorder pyruvate dehydrogenase deficiency 1 (PDP1) was first identified by us in humans and determined to be disease-causing through protein complementation assay [3]. Using the same assay conditions on cell cultures obtained from Clumber spaniels, we were able to restore pyruvate dehydrogenase activity. Molecular analysis identified the disease-causing mutation in the PDP1 gene. A genotyping test was developed, and over 100 Clumber spaniel dogs were analyzed. It was determined that the carrier frequency of the disease-causing allele was 20% of all dogs tested [4]. Our historical investigation showing over 250 years of selective breeding of a hunting dog for the elderly gentleman led us to the current molecular diagnosis, as an example of a Founder Effect, similar to that seen in humans. What was once theorized by the pioneers of population genetics can now be proven by modern-day investigation. This review combines historical descriptions of the Founder Effect as it can currently be seen by genotype analysis.

2. Mendel’s law and the founder effect

In 1865, Gregor Johann Mendel presented his results of artificial fertilization to achieve color variation with ornamental plants and this work that proposed the laws of inheritance was published the next year [5]. A generation later, when scientists had a better understanding of cells and genetic material, Mendel’s work was independently rediscovered by three botanists. In the early years of 1900, Hugo DeVries, Carl Correns, and Erich von Tschermak each confirmed Mendel’s laws of inheritance, as reviewed by Wilkie [6]. The first human disorder identified to follow Mendelian inheritance was published in 1902 by Archibald Garrod who described alkaptonuria as being inherited according to Mendelian rules. Garrod wrote of his investigation of a family, where two of five children were secreting homogentisic acid in their urine, which is pathognomonic for a diagnosis of alkaptonuria. Alkaptonuria was thought to be a peculiar disorder at the time for two reasons. First, homogentisic acid could be observed in patient’s urine from approximately 60 hours after birth and could be followed for life. Homogentisic acid causes urine to darken after exposure to air. Second, alkaptonuria did not appear to cause any detriment to the health of those with the condition but was thought to be an alternative mode of metabolism. It is now known that while the onset of clinical phenotype can be delayed in life, oxidative forms of homogentisate deposited in sclerae, cartilage, and skin can lead to early osteoarthritis and cardiac valve disease. Garrod made inquiries to several other investigators who were following alkaptonuric patients at the time, and they kindly shared their data for publication. After compiling the data for 11 families, it was found that 60% of families of children with alkaptonuria were born to first-cousin parents [7]. Garrod surmised that alkaptonuria was a congenital disorder that followed Mendelian recessive inheritance, where the rare recessive trait passed through many generations before the union of two recessive traits manifest in one zygote, as was described by Bateson in the same year [8]. The peculiar nature of alkaptonuria and the level of consanguinity in most affected families studied, allowed these early investigators to calculate the incidence of disease and postulate the mode of inheritance at a most opportunistic time when Mendel’s laws of inheritance were rediscovered.

At the time when Garrod published his findings on the incidence of alkaptonuria, and into the 1920s, the statistical study of Mendelian laws in a large population was gathering interest. Working independently, Hardy and Weinburg described similar theories that in a sexually reproducing population, the frequency of two alleles will come to equilibrium in one generation and remain unchanged provided that mating is random, the population is large and no additions to the population from outside occurs [9, 10]. The Hardy-Weinberg principle describes the relationship between allelic and genotypic frequencies in a diploid organism from one generation to another. Evolution can alter this equilibrium to include the influence of mutation, recombination, selection, and isolation. The relative frequencies of alleles in large populations can be influenced by factors that disturb equilibrium such as mutation, migration, and Darwinian selection was also considered, along with the concept of the effective population size (Ne). Ne is a mathematical representation of genetic diversity within an idealized population in comparison to a population of interest [11, 12]. In 1954, Ernst Mayr theorized that if a group of persons, which he classified as “founders”, are somehow isolated from their original large, or ancestral, population, they would abruptly undergo reduced levels of genetic variability leading to the accumulation of inbreeding within this new founder population [13]. Population geneticists have developed mathematical models to estimate how allele frequencies in a large, ancestral population can be expected to change with a sudden reduction in the population and lead to a founder effect [13]. The alleles of the founder population, with the accumulation of inbreeding, will level off to a new stable allele frequency [14]. If an unfavorable allele is in the first generation of a founder population, and this unfavorable allele has a fitness between 0.5 and 1, the allele will increase in numbers while the population doubles in size [14, 15]. Experiments have been performed in the laboratory to demonstrate rapid speciation employing an insect model to demonstrate, as recently reviewed by Haudry, Laurent, and Kapun [16].

Human population genetics typically defines the founder effect as a loss in genetic variation that can arise when a new population is derived from a small number of individuals that were segregated from a larger group. The average heterozygosity seen per locus will initially decline in the smaller, reduced group initially. Over time, new mutations will increase as the population rises. Scientists have used this theory to explain why some inherited disorders can be found at a higher frequency in a defined population than is seen in the general population. There is empirical evidence that suggests an individual in a small population that has been isolated by geography, religion, or culture, can carry an alteration to their DNA. This person is referred to as the common ancestor. After many generations, as the population expands, this variation will be seen at high frequency in the group. If the DNA variant is not disease-causing, it can be considered a polymorphism that is found at high frequency in this segregated group. If the variant is disease-causing for an autosomal recessive disorder, we will see an increase in the number of persons expressing the disorder with each successive generation, and this supports the concept of the founder effect. The founder effect has also been seen to occur naturally in animal populations. However, in many cases, it is due to humans who wish to enrich specific traits in a species of animals. Founder effect is one of the causes of the concentration of genes for specific diseases in purebred (genetic isolates) dogs [17]. Breeders of animal stock actively participate in the phenomena of heredity in real-time.

3. The Clumber spaniel

3.1 The breed

An interesting story describing the breeding of desirable traits in animals is that of the Clumber spaniel. This breed of canine is of particular fascination to the authors of this chapter as our work in the Robinson lab in 2005 on a human autosomal recessive disorder affecting pyruvate metabolism, led to the diagnosis of the same metabolic disorder in the Clumber spaniel [3, 4]. There are a few theories regarding the origin of this breed, and each is entwined in romantic, historical folklore. The most common, and rather intriguing theory begins with the aristocracy of eighteenth century France and the Noailles family. Due to the French Revolution, most of the Noailles family documents were destroyed along with many family members. Any papers that survived the Revolution were destroyed in a fire at the Louvre in May 1871 [2]. The lore suggests that in the early 1760s, Louis de Noailles (the future 4th Duc de Noailles), invited his good friend Henry Pelham-Clinton (the future 2nd Duke of Newcastle), to go hunting on his estate in France. Henry Pelham-Clinton was extremely impressed with Louis de Noailles’ hunting spaniels, particularly for their prowess in flushing game. Louis de Noailles gifted his breeding stock of Clumbers to Henry Pelham-Clinton, who brought the dogs to his Clumber Park estate in Sherwood Forest, Nottinghamshire, England in 1764. Together with the assistance of William Mansell, his gamekeeper, they further developed this breed for the “retired gentleman” or “elderly gentlemen’s hunting companion.” This breed of dog would have likely been lost forever in the chaos of the French Revolution as the French stock was wiped out [18]. The first record of Clumbers in England is seen in the 1788 painting by Sir Francis Wheatly as shown in Figure 1.

Figure 1.

Sir Francis Wheatley’s painting titled “The Return from Shooting” depicts the Duke of Newcastle on the hunt with Colonel Litchfield, his gamekeeper William Mansell, and four white with orange/lemon marked Clumber spaniels, confirming that the breed was likely in England before the of the French revolution which began on or about 1789 and ended in 1799. This picture is in the public domain and the original is in the possession of the Sheffield gallery in Sheffield, England. https://commons.wikimedia.org/w/index.php?curid=8708898

The Clumber spaniel began as a small kennel of pure-bred dogs. No genetic diversity was introduced for approximately 30 years after the breed was brought to Clumber Park and under the direction of William Mansell [18]. True to the initial selection criteria of traits, the breed is not as energetic as other hunting spaniels. The Clumbers seem to require only about an hour of activity daily. The Clumber physique is long and low, typically heavier than other Spaniels, with a white coat that can be easily seen by hunters. They continue to be developed in the United Kingdom as working gun dogs, where modest size, lack of exaggeration, good eyes, good hips, good temperament, responsiveness, athleticism, and working aptitudes are the important qualities [2]. Their personality has been described by the American Kennel Club as mellow, gentlemanly, amiable, dignified but always amusing [19]. Selective breeding of this low-energy dog has resulted in approximately 20% of current live-born Clumber spaniels being carriers for a metabolic deficiency, which results in severe exercise intolerance with lactic acidosis [4]. The metabolic deficiency in the Clumber spaniel was first reported in 1979 by Herrtage and Houlton, who described a dog that collapsed and showed extreme lactic acidosis after moderate exercise [20]. Järvinen and Sankari reported that a 3-year-old Clumber spaniel had a history of exercise intolerance since puppyhood. During exercise, the dog would quickly tire, lay down, and could not rise until resting for up to 1 hour. Post-exercise blood lactate levels were very elevated. The dog died suddenly while investigations were being performed and a defect in pyruvate dehydrogenase activity was suspected [21].

3.2 Etiology of lactic acidosis

Lactic acid is a normal product of metabolism and is utilized mainly in the liver and kidney, which express the requisite enzymes for processing, in a pathway called gluconeogenesis. During exercise, skeletal muscle produces circulating lactate which typically normalizes in time. Lactic acidosis is also a common finding in disease states when there is an increased production accompanied by decreased clearance. Metabolic lactic acidosis occurs when there is a defect in the aerobic metabolism of pyruvate. Pyruvate is naturally generated as the final product of glycolysis. Pyruvate is then used, through a series of enzymatic steps, to generate ATP. ATP is the primary energy molecule that is used by the cell for activities such as muscle contraction, synthesis of macromolecules, and maintenance of the cellular membrane. The normal fate of pyruvate is first to enter the mitochondria where it is converted to acetyl CoA by the pyruvate dehydrogenase complex. Second, acetyl CoA feeds into the citric acid cycle which generates products that drive a series of reactions of the electron transport chain to finally produce ATP. Deficits in any of the enzyme systems of these pathways will result in the accumulation of pyruvate, which in turn is reduced to lactate, thus causing lactic acidosis. The clinical features of lactic acidosis include muscle weakness, confusion, increased heart rate, metabolic acidosis with an elevated anion gap, compensatory hyperventilation due to metabolic acidosis, and in extreme cases may result in coma.

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 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.

5. Popular sire effect

The popular sire effect, also known as the popular sire syndrome, may occur when an animal with a certain trait or attribute becomes desirable. This sire will then be bred repeatedly with many females, causing a reduction of genetic diversity and allows for unconnected, undesirable genetic traits to spread rapidly. An example of selective breeding is akin to the Founder Effect that we attribute to humans. Dominant traits that have low reproductive fitness are typically removed from the gene. However, recessive traits are silently passed on until the two carriers of the recessive allele mate and have a homozygous recessive individual who is affected by a disorder. Carrier screening of the Clumber spaniel for the c.754C > T PDP1 founder mutation revealed an interesting pedigree shown below. The lines with multiple crossings highlight the complex level of consanguinity found in a subset of the breed (Figure 3). An example of a popular sire is highlighted by a gold square in Figure 3, where pedigree and genotyping analysis revealed that he was bred 11 times.

According to the Clumber Spaniel Club of America, the Clumber spaniel was one of the first breeds to be recognized by the American Kennel Club in 1884. In both the United States and the United Kingdom, it is estimated that approximately 200 Clumber puppies are registered per year. The dogs have an average lifespan of 10–12 years [23, 24]. Tests are easily available for PDP1 deficiency. The Canine Health Information Center of America reports that 362 PDP1 tests have been performed with 315 dogs being homozygous normal and 47 carriers identified [25]. Statistics from the 2018 Clumber spaniel breed health plan reports of 195 dogs tested, only 4 carriers have been identified. However, at least one parent of a breeding pair producing litters from 2015 to 2017 has not had their carrier status tested [23].

Screening of a second autosomal recessive disorder, referred to as Exercise Induced Collapse (EIC) began for Clumber spaniels in 2015. The mutation is in the dynamin 1 gene (DNM1 gene) and was originally identified in Labrador retrievers by Patterson and coworkers in 2008, where linkage analysis of affected animals led to identifying the candidate gene. Sequencing of the DNM1 gene of affected animals identified a mutation in a highly conserved region of the gene. The mutation c.767G > T alters a SmlI restriction site and a simple RFLP analysis can identify carriers and affected animals [26]. EIC is a neuromuscular disorder that presents as exercise intolerance in the absence of lactic acidosis. In contrast to the PDP1 disorder, EIC has a later onset of clinical features, at approximately 2 years of age and rarely results in early death, and does not appear to be completely penetrant. Clinical features include muscular weakness after 5–20 minutes of strenuous exercise, seen as the unsteady and uncoordinated gate of the hind limbs. Dogs typically recover from the episode within 15–30 minutes. In rare cases, EIC can progress to full-body weakness, confusion, loss of consciousness, and seizures [27]. Of the Clumbers 320 tested for EIC in the United Kingdom up to 2018, 40.8% were carriers and 4.7% were affected [23].

Pedigree analysis, linkage analysis, and modern techniques such as SNP array-based analysis of pure-breed dogs can be used to determine the coefficient of inbreeding (COI). COI refers to identical genetic information by descent and is calculated between 5 and 10 generations. It is the probability that two genes at any locus in an individual are derived from the identical diploid alleles of a previous generation [28]. For example, the COI for first cousins is 6.25%, half-siblings is 12.5%, and full siblings is 25%, provided the dam and sire do not pass through the same animal twice. For an autosomal loci, the inbreeding coefficient (F) of one individual is as shown [29]:

$$F={\left(\raisebox{1ex}{$1$}\!\left/ \!\raisebox{-1ex}{$2$}\right.\right)}^{\left(n_1+n_2+1\right)}$$E1

where n₁ and n₂ are the numbers of generations that separate the individuals of the inbred population. An example of this calculation is shown in population genetics by Johnson, Keats, and Sherman, 2019, for first cousins related through two grandparents is F = (1/2)⁵ + (1/2)⁵ = (1/2)⁴ = 1/16 or 6.25% [29].

In 2013, the COI for the Clumber spaniels in the United Kingdom was calculated to be 18.2%, which suggests low genetic diversity [30]. Repeated matings of the same dam to the same sire with complex consanguinity loops (Figure 3) are likely responsible for the loss of genetic diversity in this breed. In 2003, the UK Kennel Club recognized that the Clumber spaniel was a vulnerable British breed and was actively trying to decrease the COI by limiting the use of stud dogs and publishing the COI of breeding animals. A popular dam has a more limited effect on the COI, due to the constraints of the female reproductive cycle and litter count. With concerted effort and diligence, the COI for this breed had been reduced to 16.2% by 2017. The UK Kennel Club has been encouraging PDP1 testing results to be included on its Mate Select pages [23].

In a small founder population, such as the Clumber spaniel, random genetic drift will increase relative to selection, where beneficial alleles can become lost and harmful alleles can increase in frequency in reference to larger populations. Current technologies allow investigators to study and identify harmful alleles in founder populations. In 2023, Donner and coworkers performed SNP array analysis for over one million dogs, with 242,665 purebred dogs and 811,628 mixed-breed dogs [31]. Their data, using an unparalleled population size and following over 250 disease-associated genetic variants, show that purebred dogs indeed have a lower mean heterozygosity level than mixed-breed dogs as mathematically predicted over 100 years ago by the pioneers of population genetics. This exhaustive study reinforces the need to maintain diversity by outcrossing programs to increase heterosis (hybrid vigor introduced by mating two unrelated purebred lines that each have the desirable trait) and reducing both the use of popular sires and repeated mating with the same dam.

The genotypes of the two known heritable autosomal recessive disorders affecting the Clumber spaniel can now be determined for all breeding animals. If all carriers are restricted from mating, one would assume that the disorders would be eradicated from the breed. However, many of the dogs being bred in both the United Kingdom and United States are not being genotyped and are referred to as “Heredity Clear” meaning that the disorders were not seen in the pedigree nor within the family history of the dog. In his 1967 edition of Introduction to Quantitative Genetics, Falconer calculates the number of generations required to effect a specific change in gene frequency in an unwanted recessive allele breeding program. For example, as calculated on page 36, if the incidence of a rare autosomal recessive disorder is 1/40,000, and simply restricting the mating of homozygous affected individuals, it would take 59 generations to reduce the frequency by half [32]. Therefore, if breeders of the Clumber spaniel fail to restrict breeding carriers, it is unlikely that the disease-causing alleles will be eradicated from the breed.

To address this concern, concerted efforts of kennel clubs around the world are encouraging the publication of carrier status for breeding Clumber spaniels, which is apparently reducing the reported incidence of both PDP1 deficiency and EIC. However, due to the popular sire effect, the breed is approaching extinction if intervention is not taken. It concerns the effective population size (N_e) of the dogs. N_e is the number of breeding males and females in the population, according to the equation:

$$N_e=\frac{\left(4\times N_m\times N_f\right)}{\left(N_m+N_f\right)}$$E2

where N_m is the number of breeding males and N_f is the number of breeding females [12]. In an effective population, N_e is 100 with a sex ratio of 1 (equal amount of breeding males to females). Due to the extreme usage of a popular sire, the Clumber spaniel has very restricted inbreeding, and the Effective Population Size (N_e) is currently measured at 24.5. When N_e is lower than 100, the population size is considered critical and below 50 suggests that a breed is close to extinction [23].

6. Conclusions

The concept of purebred dogs is thought to have been created in Victorian Britain, between 1837 and 1901. However, the origin of the Clumber spaniel can be traced back to the mid-1700s, when the 4th Duc de Noailles kept a unique kennel of dogs, he kept for hunting which he shared with the 2nd Duke of Newcastle. Historical documents describe how the French Revolution caused a bottleneck event for the breed, and the Clumbers we see today are ancestors from the kennel belonging to the Duke of Newcastle at Clumber Park and carefully selected for by his gamekeeper. The Clumber was rarely seen outside grand estates until 1859 when it became fashionable to own the breed as a show dog. In the 1900s, show breeders began to favor an increased weight for the breed, and between 1908 and 2008, the weight of the show dog doubled in size [2].

Radical selection to exaggerate any features is not in the best interests of any species, as first described in the theoretical works of pioneers in the field of Population Genetics, Bottleneck, and Founder Effects such as Darwin, Mendel, Garrod, and Mayr. Recent advances in genetics and genomics allow for the identification of genetic variations. Biochemical assays allow for the correlation between mutations and how they lead to enzyme deficiencies which can then be linked to the pathogenesis of disease. Once a disease-causing mutation is known, techniques can be developed that allow for genotyping of individuals and follow the heritability of the variant within a pedigree. These technologies now allow us to prove the mathematical theories of the past. With this knowledge comes reproductive choices. Luckily, the owners and breeders of the Clumber spaniel family of canines remain lovingly invested in preserving this wonderful and royal breed and have been choosing genetic testing to reduce the coefficient of inbreeding, and through genotype analysis to identify and discourage breeding of known carriers of PDP1 and DNM1 disorders. Giving context to well-established mathematical theories, through the odyssey of the Clumber spaniel, allows for a greater understanding of population genetics (Figure 4 and Table 1).

Figure 4.

The above 7-month-old puppy was rescued from a puppy mill at 8 weeks old. He was the runt of the litter and has now been genotyped, testing positive for PDP1 deficiency. This picture is used with permission of Christy Edwards of North Carolina, USA, who is devoted to Clumber spaniel rescues.

Mating Type	Proportion of Shared Alleles	F
Parent-offspring	1/2	1/4
Full siblings	1/2	1/4
Half siblings	1/4	1/8
Uncle-niece/Aunt-nephew	1/4	1/8
First cousins	1/8	1/16
Double first cousins	1/4	1/8
Half first cousins	1/16	1/32
First cousins once removed	1/16	1/32
Second cousins	1/32	1/64
Second cousins once removed	1/64	1/128
Third cousins	1/128	1/256

Table 1.

Proportion of alleles that are shared by related individuals, adapted from Population Genetics by Johnson et al. [29].

Acknowledgments

We would like to thank Christy Edwards of North Carolina, USA, rescuer of affected Clumber spaniels bred in puppy mills, for her valuable insights and unwavering devotion to this regal breed. Her organization is called Clumber Spaniel Rescues, a Private Breed Rescue.

We also thank James Darley of Buckinghamshire, England, for personal communications, the sharing of his detailed research and historical documentation regarding the Clumber, and his devotion to the rebirth of the Royal Spaniel.

Lastly, we thank Dr. Bryant Freeman, Kansas, USA, for personal communication and the sharing of his detailed historical documentation regarding the Clumber and his devotion to the Clumber Spaniel Club of America with the title of Breed Historian.

Christy, James, and Bryant gave much-appreciated depth to our understanding of the Clumber spaniel and the 250+ year odyssey the breed has taken, leading to the small part that our group played in identifying a genetic cause for disease which is leading to increasing the health of a most Royal and Loyal breed.

Conflict of interest

The authors declare no conflict of interest.