Elaine A. Ostrander

A 120-y time series of genomes reveals the consequences of closed breeding in German Shepherd Dogs

Many contemporary breed dogs display reduced genomic health compared to mixed-breed dogs, including reduced heterozygosity and increased genetic load, likely due to strong directed breeding. Lack of historical genomes, however, has made it difficult to disentangle the timing and drivers of these declines given complex and breed-specific demographic histories, artificial selection, and crossbreeding. Here, we sequenced genomes of nine 20th-century museum specimens (1906–1993) of pedigree German Shepherd Dogs (GSDs) to directly assess the consequences of contemporary breeding practices on genomic diversity and health over time. The GSD breed offers an excellent case study, given the breed’s fluctuations in global popularity, differing selective regimes, and the GSD’s use in the establishment of other breeds, including wolfdogs. Genome-wide heterozygosity showed significant reductions after the Second World War (WWII), coincident with an increase in both the frequency of runs of homozygosity and load. We also detected repeated population bottlenecks linked to the use of popular sires throughout the 20th century. Finally, although hybridization with wolves has led to genome-wide increases in heterozygosity in wolfdog breeds derived from the GSD, nonadmixed ancestry blocks (dog or wolf) were severely depleted in diversity due to the limited number of founders involved in their establishment. Combined, our results indicate that declines in the genomic health of GSDs and related breeds occurred not at the onset of breed formation but throughout the last century, as a result of population bottlenecks associated with WWII and the repeated use of popular sires.

Genomic ancestry, behavior, and the domestication of the dog

It has been exactly 25 years since the first dog genetic maps were published and 20 years since the first dog genome sequence, a boxer, was released.The domestication of dogs was a landmark in human history, and the details of that process are key to understanding fundamental principles of evolution, domestication, and behavior.Dogs also provide the foundation for studies aimed at using canine phenotypes for studies of human health.While many millions of dogs exist as more than 350 recognized breeds, there are also an untold number of nonbreed, globally distributed village dog populations that are not under intense human selection.This special feature comprises a total of eight articles, more than half of which explore the extent and effects of genomic introgression between wolves and dogs, offering new insights and intriguing directions for future research.In addition, four studies investigate the genetic basis for different dog behaviors.The studies included in this special feature address a kaleidoscope of important issues in the evolution of dogs, the development of breeds, and the challenges faced in exploiting the novelties of canines.Each article is intended to be a launching point for discussion and experimentation.The issue highlights the convergence of multiple fields, will likely generate some controversy, and presents data that will advance the study of canines.

Genomic evidence for the Holocene codispersal of dogs and humans across Eastern Eurasia

As the first domestic species, dogs likely dispersed with different cultural groups during the Late Pleistocene and Holocene. To test this hypothesis, we analyzed 73 ancient dog genomes, including 17 newly sequenced individuals sampled from East Asia to the West Eurasian Steppe spanning nearly 10,000 years. Our results indicate correlations between the ancestry of dogs and specific ancient human populations from eastern Europe to Eastern Siberia, including Ancient Paleo-Siberians, Eastern hunter-gatherers, East Asians, and Steppe pastoralists. We also identify multiple shifts in the ancestry of dogs that coincide with specific dispersals of hunter-gatherers, farmers, and pastoralists. Combined, our results reveal the long-term and integral role that dogs played in a multitude of human societies.

Determinants of de novo mutations in extended pedigrees of 43 dog breeds

Analysis identifies germline mutation rate influenced by paternal age and CpG Islands in dogs, suggesting unique evolutionary mechanisms.

Origins and diversity of Greenland’s Qimmit revealed with genomes of ancient and modern sled dogs

The Qimmeq (Greenland sled dog) has worked continuously with the Inuit in Greenland for more than 800 years. However, they now face drastic population declines caused by climate change, urbanization, and competition from snowmobiles. This study sequenced 92 modern and ancient genomes to investigate how centuries of isolation shaped the regional Qimmeq populations and the impact of European contact. We found distinct regional populations and evidence for two migrations of dogs into Greenland with the Inuit from Canada. Furthermore, we found that there is minimal European ancestry in present day Qimmit and limited recent inbreeding despite low heterozygosity. These insights are critical for conservation efforts aimed at preserving the Qimmit amid environmental changes and cultural transitions.

Ancient dog mitogenomes support the dual dispersal of dogs and agriculture into South America

Archaeological and palaeogenomic data show that dogs were the only domestic animals introduced during the early peopling of the Americas. Hunter-gatherer groups spread quickly towards the south of the continent, but it is unclear when dogs reached Central and South America. To address this issue, we generated and analysed 70 complete mitochondrial genomes from archaeological and modern dogs ranging from Central Mexico to Central Chile and Argentina, revealing the dynamics of dog populations. Our results demonstrate that pre-contact Central and South American dogs are all assigned to a specific clade that diverged after dogs entered North America. Specifically, the divergence time between North, Central and South American dog clades is consistent with the spread of agriculture and the adoption of maize in South America between 7000 and 5000 years ago. An isolation-by-distance best characterizes how dogs expanded into South America. We identify the arrival of new lineages of dogs in post-contact South America, likely of European origin, and their legacy in modern village dogs. Interestingly, the pre-contact Mesoamerican maternal origin of the Chihuahua has persisted in some modern individuals.

Genomic analyses identify 15 risk loci and reveal HDAC2 , SOX2-OT , and IGF2BP2 in a naturally occurring canine model of gastric cancer

Gastric cancer ranks as the fifth most common human cancer worldwide and has a poor survival rate and limited treatment options. Despite the high prevalence and mortality rate, the genetic etiology is largely unknown. In dogs, a clinically and histologically similar disease disproportionately affects two breeds, the Belgian Tervuren and Belgian Sheepdog, which develop the intestinal and diffuse tumor subtypes observed in humans. We performed a Bayesian genome-wide association study and selection analyses in this naturally occurring canine model to elucidate underlying genetic risk factors for gastric cancer and identified 15 associated loci. Variant filtering revealed germline putative regulatory variants for the EPAS1 ( HIF2A ) and PTEN genes and a coding variant in CD101 . Two loci are overrepresented among cases of intestinal tumor subtype. Although closely related to Tervuren and Sheepdogs, Belgian Malinois rarely develop gastric cancer. Across-breed analyses uncovered haplotypes enriched in Malinois at SOX2-OT and IGF2BP2 that are at significantly higher frequency among genome-wide association study controls. Among Tervuren and Sheepdogs, HDAC2 putative regulatory variants were present at comparatively high frequency and were associated with risk of gastric cancer. Here, we describe a complex genetic architecture governing gastric cancer in a dog model, including genes such as PDZRN3 and KLHL29 , that have not been associated with human gastric cancer.

Genomic evidence for behavioral adaptation of herding dogs

Herding dogs exhibit a distinct constellation of behaviors marked by inherent instinct and motor skills that manipulate and guide livestock in response to instructive commands and cues. Comparison of the whole-genome sequences of herding and nonherding breeds reveals signatures of positive selection associated with pathways underlying social interaction and cognitive functions. Of the strong selective sweep signals, haplotypes within ephrin type-B receptor 1 ( EPHB1 ), which is linked to locomotor hyperactivity and spatial memory, show evidence of segregation within breed lineages for the conformation versus working lines of border collies and introgression with a genetically and geographically distant herding breed of Entlebucher mountain dogs. We show that a working line–specific haplotype of EPHB1 is associated with elevated levels of chase-bite motor patterns based on a well-validated behavior survey. These findings indicate that functional selection has shaped the genetic architecture of herding breeds, which may relate to their proficiency in addressing diverse tasks and challenges in maintaining control over the herd.

Analysis of canine gene constraint identifies new variants for orofacial clefts and stature

Dog breeding promotes within-group homogeneity through conformation to strict breed standards, while simultaneously driving between-group heterogeneity. There are over 350 recognized dog breeds that provide the foundation for investigating the genetic basis of phenotypic diversity. Typically, breed standard phenotypes such as stature, pelage, and craniofacial structure are analyzed through genetic association studies. However, such analyses are limited to assayed phenotypes only, leaving difficult-to-measure phenotypic subtleties easily overlooked. We investigated coding variation from over 2000 dogs, leading to discoveries of variants related to craniofacial morphology and stature. Breed-enriched variants were prioritized according to gene constraint, which was calculated using a mutation model derived from trinucleotide substitution probabilities. Among the newly found variants is a splice-acceptor variant in PDGFRA associated with bifid nose, a characteristic trait of Çatalburun dogs, implicating the gene's role in midline closure. Two additional LCORL variants, both associated with canine body size are also discovered: a frameshift that causes a premature stop in large breeds (>25 kg) and an intronic substitution found in small breeds (<10 kg), thus highlighting the importance of allelic heterogeneity in selection for breed traits. Most variants prioritized in this analysis are not associated with genomic signatures for breed differentiation, as these regions are enriched for constrained genes intolerant to nonsynonymous variation. This indicates trait selection in dogs is likely a balancing act between preserving essential gene functions and maximizing regulatory variation to drive phenotypic extremes.

Transcriptome Analysis of Canine Histiocytic Sarcoma Tumors and Cell Lines Reveals Multiple Targets for Therapy

Background: Histiocytic sarcoma (HS) is a highly aggressive malignancy characterized by the excessive proliferation of histiocytes in dogs and humans. A subset of dog breeds, including the Bernese Mountain Dog (BMD), show a remarkably high prevalence of HS. Previous work by us and others has identified somatic driver mutations of HS in the PTPN11 and KRAS genes that activate the MAPK pathway in about 60% of canine HS. However, no somatic driver mutations have been identified in the remaining 40%. Objectives: Our goals are to study HS in BMDs to gain insight into the molecular pathogenesis of the disease, and identify rational approaches to therapy. Methods: Here, we report our whole transcriptome analysis of 18 well-characterized BMD HS tumor tissues, as well as three HS cell lines. Results: Our analysis reveals the significant upregulation of molecular pathways involving the FOXM1, AURKB, PLK1, and E2F genes, in HS as well as hemophagocytic HS, providing new information regarding pathways that may be targeted with inhibitors. In addition, we document the expression of multiple checkpoint genes, suggesting the option of treatment with small-molecule inhibitors together with checkpoint inhibitors. Further, we show that the transcriptomes of three canine HS cell lines mirror those of canine patient tumors, further highlighting their potential use in drug discovery and efficacy studies. Finally, we demonstrate, for the first time, that aurora kinase inhibitors are effective in curtailing the growth of HS cells in vitro and show synergism with MAPK inhibition. Conclusions: This study provides the most detailed analysis of the canine HS transcriptome to date, highlighting key pathways in its pathogenesis and suggesting new avenues for both single and combination treatment strategies, which may be pertinent to the treatment of human HS.

Genomic reconstruction reveals impact of population management strategies on modern Galápagos dogs

A genotype-first approach identifies variants for orofacial clefts and other phenotypes in dogs

Dog breeding promotes within-group homogeneity through conformation to strict breed standards, and also drives between-group heterogeneity in pursuit of characteristic breed traits. There are over 350 recognized dog breeds that provide the foundation for investigating the genetic basis of phenotypic diversity. Typically, breed standard phenotypes such as stature, fur length, and craniofacial structure are analyzed in genetic association studies. However, such analyses are limited to the assayed phenotypes, leaving difficult to measure phenotypic subtleties potentially overlooked. In this study, the genotype-first approach was adapted to the dog genome to investigate coding variation from over 2000 dogs, leading to discoveries of new mutations related to craniofacial morphology and stature. Breed-enriched variants were prioritized according to gene constraint, which was calculated using a mutation model derived from trinucleotide substitution probabilities in the dog. Among the discovered variants was a splice-acceptor mutation in PDGFRA associated with bifid nose, a characteristic trait of Çatalburun dogs, implicating the gene's role in midline closure, and a frameshift mutation in LCORL associated with large canine body size, thus highlighting the importance of allelic heterogeneity in selection for breed traits. Most priority variants were not associated with genomic signatures for breed differentiation, as these regions were enriched for constrained genes intolerant to nonsynonymous variation, suggesting a model of breed phenotype diversification based on regulatory changes to essential genes. Identification of trait-associated variants in dogs informs new biological roles for genes. Improved collection of breed disease risk data, along with increased breed representation, will drive further discoveries.

Genomic analyses identify 15 susceptibility loci and revealHDAC2,SOX2-OT, andIGF2BP2in a naturally-occurring canine model of gastric cancer

SUMMARY Gastric cancer (GC) is the fifth most common human cancer worldwide, but the genetic etiology is largely unknown. We performed a Bayesian genome-wide association study and selection analyses in a naturally-occurring canine model of GC, the Belgian Tervuren and Sheepdog breeds, to elucidate underlying genetic risk factors. We identified 15 loci with over 90% predictive accuracy for the GC phenotype. Variant filtering revealed germline putative regulatory variants for the EPAS1 ( HIF2A ) and PTEN genes and a coding variant in CD101 . Although closely related to Tervuren and Sheepdogs, Belgian Malinois rarely develop GC. Across-breed analyses uncovered protective haplotypes under selection in Malinois at SOX2-OT and IGF2BP2 . Among Tervuren and Sheepdogs, HDAC2 putative regulatory variants were present at comparatively high frequency and were associated with GC. Here, we describe a complex genetic architecture governing GC in a dog model, including genes such as PDZRN3 , that have not been associated with human GC.

Co-analysis of methylation platforms for signatures of biological aging in the domestic dog reveals previously unexplored confounding factors

Chronological age reveals the number of years an individual has lived since birth. By contrast, biological age varies between individuals of the same chronological age at a rate reflective of physiological decline. Differing rates of physiological decline are related to longevity and result from genetics, environment, behavior, and disease. The creation of methylation biological age predictors is a long-standing challenge in aging research due to the lack of individual pre-mortem longevity data. The consistent differences in longevity between domestic dog breeds enable the construction of biological age estimators which can, in turn, be contrasted with methylation measurements to elucidate mechanisms of biological aging. We draw on three flagship methylation studies using distinct measurement platforms and tissues to assess the feasibility of creating biological age methylation clocks in the dog. We expand epigenetic clock building strategies to accommodate phylogenetic relationships between individuals, thus controlling for the use of breed standard metrics. We observe that biological age methylation clocks are affected by population stratification and require heavy parameterization to achieve effective predictions. Finally, we observe that methylation-related markers reflecting biological age signals are rare and do not colocalize between datasets.

Germline Sequencing of DNA Damage Repair Genes in Two Hereditary Prostate Cancer Cohorts Reveals New Disease Risk-Associated Gene Variants

Rare, inherited variants in DNA damage repair (DDR) genes have a recognised role in prostate cancer (PrCa) susceptibility. In addition, these genes are therapeutically targetable. While rare variants are informing clinical management in other common cancers, defining the rare disease-associated variants in PrCa has been challenging. Here, whole-genome and -exome sequencing data from two independent, high-risk Australian and North American familial PrCa datasets were interrogated for novel DDR risk variants. Rare DDR gene variants (predicted to be damaging and present in two or more family members) were identified and subsequently genotyped in 1963 individuals (700 familial and 459 sporadic PrCa cases, 482 unaffected relatives, and 322 screened controls), and association analyses accounting for relatedness (MQLS) undertaken. In the combined datasets, rare ERCC3 (rs145201970, p = 2.57 × 10−4) and BRIP1 (rs4988345, p = 0.025) variants were significantly associated with PrCa risk. A PARP2 (rs200603922, p = 0.028) variant in the Australian dataset and a MUTYH (rs36053993, p = 0.031) variant in the North American dataset were also associated with risk. Evaluation of clinicopathological characteristics provided no evidence for a younger age or higher-grade disease at diagnosis in variant carriers, which should be taken into consideration when determining genetic screening eligibility criteria for targeted, gene-based treatments in the future. This study adds valuable knowledge to our understanding of PrCa-associated DDR genes, which will underpin effective clinical screening and treatment strategies.

Multiple ancestries and shared gene flow among modern livestock guarding dogs

Livestock guarding dogs (LGDs) have been used to protect livestock for millennia. While previous works suggested a single origin of modern LGDs, the degree and source of shared ancestry have not been tested. To address this, we generated genome-wide SNP data from 304 LGDs and combined it with public genomic data from 2,183 modern and 22 ancient dogs. Our findings reveal shared ancestry and extensive gene flow among modern LGD breeds which we attribute to historical livestock migrations. Additionally, admixture between LGDs and free-ranging dogs argues against reproductive isolation as a core mechanism for maintaining the specialized skills of LGDs. Finally, we identify two lineages within modern LGDs and uncover multiple ancestries tracing back to distinct Eurasian ancient dogs, concordant with the absence of a single ancestor. Overall, our work explores the complex evolutionary history of LGDs, offering valuable insights into how human and livestock co-migrations shaped this functional group.

JUN 2024OstranderGenetics

Dogs and their genes: what ever will they think of next?

The Edward Novitski Prize recognizes creativity and intellectual ingenuity in the solution of problems in genetics research. The prize honors scientific experimental work—either a single experimental accomplishment or a body of work. Ostrander is recognized for work developing the domestic dog as an experimental system for solving fundamental biological problems and identifying genetic sequences of relevance to human health and disease. Including work on disease and behavioral health, Ostrander has shown a dedication to creative methods for understanding canine genetics and the value of translating research organisms to human genetics.

Determinants of de novo mutations in extended pedigrees of 43 dog breeds

Intensive breeding of dogs has had dramatic effects on genetic variants underlying phenotypes. To investigate whether this also affected mutation rates, we deep-sequenced pedigrees from 43 different dog breeds representing 404 trios. We find that the mutation rate is remarkably stable across breeds and is predominantly influenced by variation in parental ages. The effect of paternal age per year on mutation rates is approximately 1.5 times greater in dogs than humans, suggesting that the elevated yearly mutation rate in dogs is only partially attributed to earlier reproduction. While there is no significant effect of breeds on the overall mutation rate, larger breeds accumulate proportionally more mutations earlier in development than small breeds. Interestingly, we find a 2.6 times greater mutation rate in CG Islands (CGIs) compared to the remaining genome in dogs, unlike humans, where there is no difference. Our estimated rate of mutation by recombination in dogs is more than 10 times larger than estimates in humans. We ascribe these to the fact that canids have lost PRDM9-directed recombination and draw away recombination from CGIs. In conclusion, our study sheds light on stability of mutation processes and disparities in mutation accumulation rates reflecting the influence of differences in growth patterns among breeds, and the impact of PRDM9 gene loss on the de novo mutations of canids.

Large-scale genomic analysis of the domestic dog informs biological discovery

Recent advances in genomics, coupled with a unique population structure and remarkable levels of variation, have propelled the domestic dog to new levels as a system for understanding fundamental principles in mammalian biology. Central to this advance are more than 350 recognized breeds, each a closed population that has undergone selection for unique features. Genetic variation in the domestic dog is particularly well characterized compared with other domestic mammals, with almost 3000 high-coverage genomes publicly available. Importantly, as the number of sequenced genomes increases, new avenues for analysis are becoming available. Herein, we discuss recent discoveries in canine genomics regarding behavior, morphology, and disease susceptibility. We explore the limitations of current data sets for variant interpretation, tradeoffs between sequencing strategies, and the burgeoning role of long-read genomes for capturing structural variants. In addition, we consider how large-scale collections of whole-genome sequence data drive rare variant discovery and assess the geographic distribution of canine diversity, which identifies Asia as a major source of missing variation. Finally, we review recent comparative genomic analyses that will facilitate annotation of the noncoding genome in dogs.

Genome-wide analyses reveals an association between invasive urothelial carcinoma in the Shetland sheepdog and NIPAL1

Naturally occurring canine invasive urinary carcinoma (iUC) closely resembles human muscle invasive bladder cancer in terms of histopathology, metastases, response to therapy, and low survival rate. The heterogeneous nature of the disease has led to the association of large numbers of risk loci in humans, however most are of small effect. There exists a need for new and accurate animal models of invasive bladder cancer. In dogs, distinct breeds show markedly different rates of iUC, thus presenting an opportunity to identify additional risk factors and overcome the locus heterogeneity encountered in human mapping studies. In the association study presented here, inclusive of 100 Shetland sheepdogs and 58 dogs of other breeds, we identify a homozygous protein altering point mutation within the NIPAL1 gene which increases risk by eight-fold (OR = 8.42, CI = 3.12–22.71), accounting for nearly 30% of iUC risk in the Shetland sheepdog. Inclusion of six additional loci accounts for most of the disease risk in the breed and explains nearly 75% of the phenotypes in this study. When combined with sequence data from tumors, we show that variation in the MAPK signaling pathway is an overarching cause of iUC susceptibility in dogs.