Genetic diversity in contemporary Samoyed
The objective of this study was to characterize an autosomal recessive mutation that appeared in Samoyed and to determine how genetic diversity and inbreeding may have contributed to its origin, spread, and potential effect on existing diversity. Therefore, genetic diversity in contemporary Samoyed was first evaluated. Genetic diversity can be measured from pedigrees, but if mating is non-random, their accuracy and quantity must be high [28]. Pedigrees used for genetic diversity must also include all founders that contributed to the breed [29]. Therefore, it is increasingly common to include both deep pedigree and DNA analysis in studies involving genetic diversity. Extensive pedigrees and large SNP arrays have been used to determine genetic diversity and genome substructure in dog breeds such as the Bull Mastiff [30]. One problem with large SNP arrays is to find ways to present complex data sets in a simple manner [31]. Data from STRs (microsatellites, single sequence repeats) is easily understood and has been used in combination with pedigrees in genetic analyses of breeds such as Standard Poodles [14] and Italian Greyhound [16]. STRs are also considered still informative in plant genetics [32] and research on certain meat breeds of cattle found STRs to be equivalent to pedigrees [33]. STRs were used in-lieu of pedigrees in genetic studies of the Bulldog [13] and several other breeds.14 STRs are also economical and can work with limited amounts of DNA and even poor quality DNA. Unlike STRs, SNPs cannot interrogate highly polymorphic regions such as the DLA. Therefore, genetic diversity in the present study was determined with small panels of 33 STR loci on 25/38 autosomes and 7 STRs defining the DLA class I and II regions on CFA12.
It is uncertain how many dogs constituted the founding population for the Samoyed, although an analysis of pedigrees from dogs registered in the USA from 1990 to 1999 by Bell (2002)Footnote 18 showed an increasing number of the same individuals in earlier generations. This suggested a small founder population. However, an analysis of a large number of pedigrees as part of this same study showed that the average inbreeding coefficient of contemporary Samoyed was 9.94% +/− 7.64 and it was concluded that “that Samoyed has acceptable overall breed-wide diversity.”18 Sorsa,Footnote 19 using SNP arrays (MyDogDNA pilot analysis), found that the genetic diversity of Samoyed (also known as Bjelkier in Europe) was above the median of 3000 dogs of all breeds in their database and concluded that “even though the population size of less than 50 dogs (used in the study) is very small, it gives an initial indication that the active Samoyed breeders and owners would have succeeded in their systematic efforts for preserving diversity.”
The most extensive population and genetic study of the Samoyed was reported by the UK kennel club for the period between 1980 and 2014.10 There were about 200 Samoyed registered in the UK in 1980, but this rapidly increased to a peak of over 1200 dogs by 1995. The breed appeared to lose popularity after this time and only about 300 Samoyed were registered in 2014. The population increase was closely linked to the number of puppies produced each year by a single sire.10 A single sire averaged 2.32 puppies in 1980, 10.56 offspring in 1994 and that dropped back to 7.05 in 2014. An observed and expected inbreeding coefficient was calculated and graphed for the period 1980–2014.10 The inbreeding coefficient is the probability that two alleles at a given gene locus in random members of a population are identical. In 1980, the observed inbreeding coefficient was 0.022, while the expected inbreeding coefficient was around 0.015.10 The difference was 0.007, or a 0.7% rise in identical alleles in the population prior to the population bubble starting in 1980. In contrast, the observed inbreeding coefficient at the peak of the population in 2001 was 0.11 and the expected inbreeding coefficient to 0.06, a calculated difference of 0.05. Therefore, allele sharing based on these figures increased by 4.3% (0.05–0.007) from 1989 to 2001, while the observed coefficient of inbreeding dropped to 0.08 in 2014 as the population numbers declined. Using the figures of this study,10 the expected coefficient of inbreeding at 2014 was around 0.06, a difference of 0.02, indicating a trend towards more random breeding. Overall, allele sharing among UK Samoyed increased from 0.7% in 1980 to 5% in 2001 and back to 0.2% in 2014. The author of UK kennel club survey concluded: “As with most breeds, the rate of inbreeding was at its highest in this breed in the 1980s and 1990s. This represents a ‘genetic bottleneck’, with genetic variation lost from the population. However, since 2000 the rate of inbreeding has slowed and even declined slightly, implying maintenance and even some replenishment of genetic diversity (possibly through the use of imported animals).”
10 There are two problems with this conclusion. Inbreeding and genetic variation (diversity) are different things. It is possible to expand a portion of the population by rapid inbreeding without losing any original genetic diversity. Inbreeding can be reversed if even remnants of original diversity survive, but genetic diversity that is permanently lost through inbreeding can never be replaced from within the population. The conclusion of the UK Kennel Club study was also misleading because it implies that allele sharing at each locus is not a problem in the breed. Based on DNA testing, the average percentage of homozygous alleles across the 33 autosomal STR loci in contemporary Samoyed was 39% (SD = 9.5%), while the average percentage of homozygous alleles was 54% (SD = 38%) for the 7 STR loci in the DLA class I and II regions. Therefore, allele sharing in Samoyed is greater than the percentages indicated by this 1980–2014 population study by the UK Kennel Club. Given a high level of pre-existing allele sharing, the loss of even small amounts of heterozygosity in such population bubbles can have a dramatic effect. Such bouts of inbreeding are also times when new mutations occur and/or when existing deleterious mutations are amplified to clinical levels. It is possible, given the timelines that the causative mutation for ARAI in Samoyed occurred during this population bubble.
The increased inbreeding observed during the 1980s and 1990s is typical of what occurs when a breed becomes popular and the need for puppies and their value greatly increases. Professional breeders usually blame commercial breeders for the great increase in inbred dogs during such periods, and there is a measure of truth to this belief. This is exemplified by what happens when popularity declines. Commercial breeders leave the market and professional breeders tend to return to more random breeding practices.
The same UK Kennel club survey also provided an estimate of the effective population size for Samoyed of 64.7 dogs.10 Effective population size, founder equivalents and founder genome equivalents are related terms that usually refer to the “number of equally contributing (randomly breeding) founders that would be expected to produce (maintain) the same genetic diversity as in the population under study [29].” The question is whether 64.7 Samoyed, possessing the present spectrum of genetic diversity and freely breeding, would be sufficient to maintain the current level of genetic diversity. A minimum number comes from the 50/500 rule proposed by Franklin in 1980 [34]. The “50” part of the 50/500 rule states that inbred populations with an effective population size under 50 are at immediate risk of extinction if the population size should fall below 500 individuals. Breeding within such a small inbred population, whether it is random or nonrandom, can quickly force a small population into a downward spiral of lost diversity (i.e., an extinction vortex). In purebred dog terms, a genetic bottleneck such as a popular sire effect can lead to a decrease in effective population size, while a catastrophic event such as a world war or loss of popularity can reduce the population size to fewer than 500 dogs. The 50/500 rule has more application to wild populations subject to survival of the fittest, while most dog breeds can be sustained by considerable human intervention and sophisticated veterinary care, an example being the Bulldog [13]. Nonetheless, the world-wide number of Samoyed is well over 500 dogs and is presumably sufficient to maintain existing genetic diversity if properly managed to avoid further cycles of intense inbreeding.
The present studies confirm and expand upon the findings of others regarding genetic diversity in the Samoyed. One hundred eighty-two dogs from different geographic regions of the world should provide a reliable measure, based on other breeds studied by our group, of the incidence of all major and most minor alleles present at each of the 33 autosomal STR loci and comparative incidence of DLA class I and II haplotypes.14 The amount of genetic diversity found in contemporary Samoyed was greater than our group found in breeds such as Flat-coated retrievers, Doberman, and Bulldog; similar to the Alaskan Klee Kai, Biewer, and Black Russian Terrier; and less than Standard and Miniature Poodle, Havanese, Akita, Golden Retriever, and Italian Greyhound.14 There was evidence for some geographic differentiation of European and Australian dogs but the numbers, especially of Australian dogs were too small to confirm this finding. However, this differentiation was only slight and far less identifiable than between Japanese and American Akita as tested by our laboratoryFootnote 20 or American and European Italian Greyhound [16]. The geographic differentiation was more like that demonstrated for English and American Standard Poodle [15]. The genetic homogeneity of Samoyed from disparate parts of the world was somewhat surprising given the fact that the breed started in several different countries at about the same time. This suggests that there has been a great deal of international exchange of Samoyed over the last century.
Allele frequencies at each of the 33 autosomal STR loci indicated that 70–90% of the population was closely related to each other. Without accurate information on breed founders, it is not possible to say whether there were few founders at the start or that many founders were lost as the breed evolved over the last century. The DLA haplotypes confirmed that the contemporary Samoyed has been heavily selected for two lineages. The DLA region is in strong linkage disequilibrium and each class I and II haplotype is inherited largely unchanged over a long period of time from sire and dam. About 90% of all Samoyed tested shared three class I and four class II haplotypes. One class I (1152) and one class II (2053) haplotypes were found in over 50% of the dogs. The major 1152 haplotype along with three less common haplotypes were unique to the breed among those identified to date by our group.14 The major 2053 haplotype was also found in other breeds, while the Samoyed possess three unique class II haplotypes. These unique and/or major haplotypes are most certain to belong to founder dogs that had features strongly engrained in the breed standard and have been diligently maintained. A standard genetic assessment of the 7 STR loci associated with these haplotypes indicates that these haplotypes, although highly unbalanced in proportion, are largely in breed-wide equilibrium.
Incidence of mutation responsible for ARAI
It is uncertain when the first case of enamel hypoplasia occurred in the breed, although some recall possible cases as far back as 1985. Based on the oldest age of affected dogs, the causative mutation has been present for several generations; the oldest affected dog was 11 years of age and assuming the parents were 4–5 years old at its birth. Both parents also had to be either heterozygous or homozygous for the mutation, pushing the timeline back several more generations. Although the mutation may be old, the occurrence of diseased dogs has been apparently increasing of late. This suggests that the mutation has been under recent positive selection, most likely in association with a desired trait. There are rumors that an affected popular sire may be involved, but this was not pursued in the present study. However, this is where pedigrees associated with DNA testing could be highly informative as to the precise origin of the mutation and the cause for its recent spread. Detective work of this type was used to trace the origins of sebaceous adenitis and Addison’s disease in the Standard Poodle [14]. The exact incidence of affected dogs is also not known. The incidence of carriers among the healthy dogs that were tested was around 12%, which would make the incidence of affected dogs for an autosomal recessive disease 0.12 × 0.12 × 0.25 = 0.0036 or 3.6/1000. If this figure is correct, the incidence of affected dogs is below the incidence of 1–2% or greater when deleterious autosomal recessive traits become of concern in other breeds.
Genetic basis for ARAI in Samoyed
Enamel formation is a complex process involving many genes working in unison and at various stages of tooth development. Wright and colleagues [35] listed 91 conditions in Online Mendelian Inheritance in Man as having an enamel phenotype, and of those, 71 have a known molecular etiology or linked genetic loci. The complexity of enamel formation allows for mutations in many different genes to cause a similar defect. Therefore, it is not surprising that enamel hypoplasia has been observed in many breeds, although only one other mutation causing a heritable enamel hypoplasia has been characterized to date, an ARAI involving the enamelin gene (ENAM) in Italian Greyhounds [17]. One reason for this has been the relatively recent growth of the specialty of veterinary dentistry and the previous tendency to dismiss such cases as due to non-heritable causes such as canine distemper, high fevers, drugs or traumas occurring during puppy-hood [36]. The enamel dysplasia described up to this time has also been relatively mild and easily confused with plaque build-up and tooth discoloration in dogs. The enamel hypoplasia in Italian Greyhound is much more common, but also less severe [17]. An even milder enamel hypoplasia that appears to be heritable has been reported in the Standard Poodle [37]. The complexity of genes involved in enamel formation, and the large possible numbers of mutations affecting enamel is reminiscent of the large number of genes associated with vision and the many genetic causes of progressive retinal atrophy [38].
Two potential deleterious mutations in SCL24A on CFA8 and in strong linkage disequilibrium were ultimately identified in ARAI affected dogs, an asynonymous change (C to T) in exon 12 changing amino acid proline to leucine and a 21 bp duplication in exon 17. The leucine to proline change in exon 12 has been reported in GenBank in golden snub-nosed monkey (XP_010382802, XP_010382799, XP_010382800) and camel (XP_006184249, XP_006184251) and was assumed to be a polymorphism present in normal animals and not deleterious. The insertion in exon 17 that caused a 21 bp insertion was in the terminal transmembrane region of SLC24A4 and would be more likely to inhibit function of the 4SCL24A4 protein, which transports one Ca++ and K+ ion in exchange for four Na + ions [41]. A duplication of this size in terminal exon 17 would presumably change the size of an internal-membrane portion of the protein.
The large run of homozygosity (selective sweep) containing these SCL24A4 mutations was highly conserved among all the affected Samoyed tested, indicating that the region possesses many genes and gene polymorphisms that define the breed phenotype in some manner. It is also likely that a polymorphism that was deemed favorable to the breed, most likely involving a conformational trait, also occurred in or near this same region and that it was amplified as well. The number of these selective sweeps varies greatly in number and size between breeds. However, sweeps become larger and more numerous as the phenotype deviates more and more from the ancestral dog. As such, the Bulldog and Bull Mastiff have numerous large selective sweeps [13, 39], whiles the Standard Poodle, with the same level of SNP homozygosity, has fewer and smaller selective sweeps in its genome [13, 40]. The presence of numerous selective sweeps, when coupled with frequent changes in desired show traits and artificial positive selection for those phenotypes, are significant factors behind the high incidence of genetic disorders in pure breeds of animals. Dog breeds have undergone and continue to undergo the most phenotypic change, also have among the highest incidence of heritable disease [13]. If the Samoyed should continue to lose genetic diversity and be subjected to continued bouts of conformational change, other simple genetic diseases will occur. As a dog breed becomes more inbred the incidence of complex genetic traits will also rise [13, 14, 16].
Comparison of Samoyed ARAI with SLC24A4 ARAI in people
SLC24A4 has only been recently identified as a cause of amelogenesis imperfecta (AI) in humans. Parry and colleagues [41] identified a missense mutation in the ion binding site of SLC24A4 in a family with hypocalcified teeth. Seyman et al. [42] described a 10 kb deletion in covering exons 15, 16 and most of 17 in a family with brown discoloration of the teeth. A third mutation was also identified as a missense T > G mutation in SLC24A4 causing a leucine to arginine switch at position 436 and closely resembles the clinical appearance of the enamel hypoplasia in Samoyed [43] (Fig. 10).
Strategy for control of ARAI in Samoyed
What should breeders do when a genetic mutation such as this occurs in their breed? The answer depends on how much genetic diversity will be lost in the attempt to eliminate it from the breed. In the case of Pug Dog Encephalitis, the recessive heritable associations to risk for the disease were present in one third of the dogs and the breed was limited in diversity [44]. Therefore, a decision was made to breed away from homozygotes, while maintaining the trait in a heterozygous state. The outcome of eliminating the SCL24A allele was tested in the present study by creating test populations containing a known proportion of carrier dogs, and then measuring genetic diversity before and after the carrier population is removed. The results of such testing indicated that Samoyed breeders could easily eliminate the trait without loss of genetic diversity due to the low incidence of the mutation at present time.