A cautionary tale of low-pass sequencing and imputation with respect to haplotype accuracy.

BACKGROUND: Low-pass whole-genome sequencing and imputation offer significant cost savings, enabling substantial increases in sample size and statistical power. This approach is particularly promising in livestock breeding, providing an affordable means of screening individuals for deleterious alleles or calculating genomic breeding values. Consequently, it may also be of value in companion animal genomics to support pedigree breeding. We sought to evaluate in dogs the impact of low coverage sequencing and reference-guided imputation on genotype concordance and association analyses. RESULTS: DNA isolated from saliva of 30 Labrador retrievers was sequenced at low (0.9X and 3.8X) and high (43.5X) coverage, and down-sampled from 43.5X to 9.6X and 17.4X. Genotype imputation was performed using a diverse reference panel (1021 dogs), and two subsets of the former panel (256 dogs each) where one had an excess of Labrador retrievers relative to other breeds. We observed little difference in imputed genotype concordance between reference panels. Association analyses for a locus acting as a disease proxy were performed using single-marker (GEMMA) and haplotype-based (XP-EHH) tests. GEMMA results were highly correlated (r ≥ 0.97) between 43.5X and ≥ 3.8X depths of coverage, while for 0.9X the correlation was lower (r ≤ 0.8). XP-EHH results were less well correlated, with r ranging from 0.58 (0.9X) to 0.88 (17.4X). Across a random sample of 10,000 genomic regions averaging 17 kb in size, we observed a median of three haplotypes per dog across the sequencing depths, with 5% of the regions returning more than eight haplotypes. Inspection of one such region revealed genotype and phasing inconsistencies across sequencing depths. CONCLUSIONS: We demonstrate that saliva-derived canine DNA is suitable for whole-genome sequencing, highlighting the feasibility of client-based sampling. Low-pass sequencing and imputation require caution as incorrect allele assignments result when the subject possesses alleles that are absent in the reference panel. Larger panels have the capacity for greater allelic diversity, which should reduce the potential for imputation error. Although low-pass sequencing can accurately impute allele dosage, we highlight issues with phasing accuracy that impact haplotype-based analyses. Consequently, if accurately phased genotypes are required for analyses, we advocate sequencing at high depth (> 20X).

Identification of Novel Coloboma Candidate Genes through Conserved Gene Expression Analyses across Four Vertebrate Species.

Ocular coloboma (OC) is a failure of complete optic fissure closure during embryonic development and presents as a tissue defect along the proximal-distal axis of the ventral eye. It is classed as part of the clinical spectrum of structural eye malformations with microphthalmia and anophthalmia, collectively abbreviated to MAC. Despite deliberate attempts to identify causative variants in MAC, many patients remain without a genetic diagnosis. To reveal potential candidate genes, we utilised transcriptomes experimentally generated from embryonic eye tissues derived from humans, mice, zebrafish, and chicken at stages coincident with optic fissure closure. Our in-silico analyses found 10 genes with optic fissure-specific enriched expression: ALDH1A3, BMPR1B, EMX2, EPHB3, NID1, NTN1, PAX2, SMOC1, TENM3, and VAX1. In situ hybridization revealed that all 10 genes were broadly expressed ventrally in the developing eye but that only PAX2 and NTN1 were expressed in cells at the edges of the optic fissure margin. Of these conserved optic fissure genes, EMX2, NID1, and EPHB3 have not previously been associated with human MAC cases. Targeted genetic manipulation in zebrafish embryos using CRISPR/Cas9 caused the developmental MAC phenotype for emx2 and ephb3. We analysed available whole genome sequencing datasets from MAC patients and identified a range of variants with plausible causality. In combination, our data suggest that expression of genes involved in ventral eye development is conserved across a range of vertebrate species and that EMX2, NID1, and EPHB3 are candidate loci that warrant further functional analysis in the context of MAC and should be considered for sequencing in cohorts of patients with structural eye malformations.

Identification of Genetic Risk Factors for Monogenic and Complex Canine Diseases.

Advances in DNA sequencing and other technologies have greatly facilitated the identification of genetic risk factors for inherited diseases in dogs. We review recent technological developments based on selected examples from canine disease genetics. The identification of disease-causing variants in dogs with monogenic diseases may become a widely employed diagnostic approach in clinical veterinary medicine in the not-too-distant future. Diseases with complex modes of inheritance continue to pose challenges to researchers but have also become much more tangible than in the past. In addition to strategies for identifying genetic risk factors, we provide some thoughts on the interpretation of sequence variants that are largely inspired by developments in human clinical genetics.

The impact of the COVID-19 pandemic on a cohort of Labrador retrievers in England.

BACKGROUND: The COVID-19 pandemic is likely to have affected the welfare and health of dogs due to surges in adoptions and purchases, changes in the physical and mental health and financial status of dog owners, changes in dogs' lifestyle and routines and limited access to veterinary care. The aims of this study were to investigate whether COVID-19 restrictions were associated with differences in Labrador retrievers' lifestyle, routine care, insurance status, illness incidence or veterinary attendance with an illness, who were living in England and enrolled in Dogslife, an owner-based cohort study. Longitudinal questionnaire data from Dogslife that was relevant to the dates between the 23rd of March and the 4th of July 2020, during COVID-19 restrictions in England, were compared to data between the same dates in previous years from 2011 to 2019 using mixed regression models and adjusted chi-squared tests. RESULTS: Compared with previous years (March 23rd to July 4th, 2010 to 2019), the COVID-19 restrictions study period (March 23rd to July 4th 2020) was associated with owners reporting increases in their dogs' exercise and worming and decreases in insurance, titbit-feeding and vaccination. Odds of owners reporting that their dogs had an episode of coughing (0.20, 95% CI: 0.04-0.92) and that they took their dogs to a veterinarian with an episode of any illness (0.58, 95% CI: 0.45-0.76) were lower during the COVID-19 restrictions compared to before. During the restrictions period, owners were less likely to report that they took their dogs to a veterinarian with certain other illnesses, compared to before this period. CONCLUSIONS: Dogslife provided a unique opportunity to study prospective questionnaire data from owners already enrolled on a longitudinal cohort study. This approach minimised bias associated with recalling events prior to the pandemic and allowed a wider population of dogs to be studied than is available from primary care data. Distinctive insights into owners' decision making about their dogs' healthcare were offered. There are clear implications of the COVID-19 pandemic and associated restrictions for the lifestyle, care and health of dogs.

More Than a Moggy; A Population Genetics Analysis of the United Kingdom's Non-Pedigree Cats.

The domestic cat is one of the most popular pets in the world. It is estimated that 89-92% of domestic cats in the UK are non-pedigree Domestic shorthair (DSH), Domestic longhair (DLH), or Domestic semi-longhair cats (DSLH). Despite their popularity, little is known of the UK non-pedigree cats' population structure and breeding dynamics. Using a custom designed single nucleotide variant (SNV) array, this study investigated the population genetics of 1344 UK cats. Principal components analysis (PCA) and fastSTRUCTURE analysis verified that the UK's DSH, DLH, and DSLH cats are random-bred, rather than admixed, mix breed, or crossbred. In contrast to pedigree cats, the linkage disequilibrium of these random-bred cats was least extensive and decayed rapidly. Homozygosity by descent (HBD) analysis showed the majority of non-pedigree cats had proportionally less of their genome in HBD segments compared to pedigree cats, and that these segments were older. Together, these findings suggest that the DSH, DLH, and DSLH cats should be considered as a population of random-bred cats rather than a crossbred or pedigree-admixed cat. Unexpectedly, 19% of random-bred cat genomes displayed a higher proportion of HBD segments associated with more recent inbreeding events. Therefore, while non-pedigree cats as a whole are genetically diverse, they are not impervious to inbreeding and its health risks.

Surveillance of a vomiting outbreak in dogs in the UK using owner-derived and internet search data.

BACKGROUND: In early 2020, the Small Animal Veterinary Surveillance Network reported evidence of an outbreak of acute prolific vomiting in dogs in the UK. The aims of this study were to investigate whether there was evidence for a vomiting outbreak in Dogslife and Google Trends data and to describe its characteristics. METHODS: Incidence of Dogslife vomiting reports and the Google search index for 'dog vomiting' and 'puppy vomiting' between December 2019 and March 2020 was compared to the respective data from the same months in previous years. Risks for dogs vomiting and factors influencing veterinary attendance in Dogslife were identified using multivariable logistic regression. RESULTS: This study confirmed a vomiting outbreak was evident in UK dogs between December 2019 and March 2020 using data from Dogslife and Google Trends. The odds of a vomiting incident being reported to Dogslife was 1.51 (95% CI: 1.24-1.84) in comparison to previous years. Dogslife data identified differences in owner-decision making when seeking veterinary attention and identified factors associated with dogs at higher odds of experiencing a vomiting episode. CONCLUSION: Owner-derived data including questionnaires and internet search queries should be considered a valid, valuable source of information for veterinary population health surveillance.

A Highly Conserved Shh Enhancer Coordinates Hypothalamic and Craniofacial Development.

Enhancers that are conserved deep in evolutionary time regulate characteristics held in common across taxonomic classes. Here, deletion of the highly conserved Shh enhancer SBE2 (Shh brain enhancer 2) in mouse markedly reduced Shh expression within the embryonic brain specifically in the rostral diencephalon; however, no abnormal anatomical phenotype was observed. Secondary enhancer activity was subsequently identified which likely mediates low levels of expression. In contrast, when crossing the SBE2 deletion with the Shh null allele, brain and craniofacial development were disrupted; thus, linking SBE2 regulated Shh expression to multiple defects and further enabling the study of the effects of differing levels of Shh on embryogenesis. Development of the hypothalamus, derived from the rostral diencephalon, was disrupted along both the anterior-posterior (AP) and the dorsal-ventral (DV) axes. Expression of DV patterning genes and subsequent neuronal population induction were particularly sensitive to Shh expression levels, demonstrating a novel morphogenic context for Shh. The role of SBE2, which is highlighted by DV gene expression, is to step-up expression of Shh above the minimal activity of the second enhancer, ensuring the necessary levels of Shh in a regional-specific manner. We also show that low Shh levels in the diencephalon disrupted neighbouring craniofacial development, including mediolateral patterning of the bones along the cranial floor and viscerocranium. Thus, SBE2 contributes to hypothalamic morphogenesis and ensures there is coordination with the formation of the adjacent midline cranial bones that subsequently protect the neural tissue.

From head to hind: Elucidating function through contrasting morphometrics of ancient and modern pedigree dogs.

Used together, caliper- and geometric-based morphometric analyses provide complimentary approaches to classifying form and function of archaeozoological remains. Here we apply these analytical tools to the skeletal remains of an ancient male dog unearthed from a rural farm settlement of Roman date near present day Warmington, United Kingdom. Our comparisons of the Warmington Roman dog against the morphological characteristics of modern dog breeds enabled us to establish the former's size and shape. It was of medium stature. Analysis of viscerocrania and neurocrania indicate it falls within the meso- to dolichocephalic rankings of modern dogs. The neurocranium shape and the dimensions of its long bones strongly suggest that the Warmington dog shares similarities to modern sight hounds. Historically sight hounds were bred for speed, as necessitated of a hunter that runs down small prey. Our analysis suggests that the Warmington dog was likely bred for, or derived from, Roman hunting stock. By revealing the Warmington Roman dog's form from cranial and postcranial analyses, we shed light on Roman life in one of the furthest outposts of the Roman Empire.

Characterisation of a second gain of function EDAR variant, encoding EDAR380R, in East Asia.

Ectodysplasin A1 receptor (EDAR) is a TNF receptor family member with roles in the development and growth of hair, teeth and glands. A derived allele of EDAR, single-nucleotide variant rs3827760, encodes EDAR:p.(Val370Ala), a receptor with more potent signalling effects than the ancestral EDAR370Val. This allele of rs3827760 is at very high frequency in modern East Asian and Native American populations as a result of ancient positive selection and has been associated with straighter, thicker hair fibres, alteration of tooth and ear shape, reduced chin protrusion and increased fingertip sweat gland density. Here we report the characterisation of another SNV in EDAR, rs146567337, encoding EDAR:p.(Ser380Arg). The derived allele of this SNV is at its highest global frequency, of up to 5%, in populations of southern China, Vietnam, the Philippines, Malaysia and Indonesia. Using haplotype analyses, we find that the rs3827760 and rs146567337 SNVs arose on distinct haplotypes and that rs146567337 does not show the same signs of positive selection as rs3827760. From functional studies in cultured cells, we find that EDAR:p.(Ser380Arg) displays increased EDAR signalling output, at a similar level to that of EDAR:p.(Val370Ala). The existence of a second SNV with partly overlapping geographic distribution, the same in vitro functional effect and similar evolutionary age as the derived allele of rs3827760, but of independent origin and not exhibiting the same signs of strong selection, suggests a northern focus of positive selection on EDAR function in East Asia.

Is it time to stop sweeping data cleaning under the carpet? A novel algorithm for outlier management in growth data.

All data are prone to error and require data cleaning prior to analysis. An important example is longitudinal growth data, for which there are no universally agreed standard methods for identifying and removing implausible values and many existing methods have limitations that restrict their usage across different domains. A decision-making algorithm that modified or deleted growth measurements based on a combination of pre-defined cut-offs and logic rules was designed. Five data cleaning methods for growth were tested with and without the addition of the algorithm and applied to five different longitudinal growth datasets: four uncleaned canine weight or height datasets and one pre-cleaned human weight dataset with randomly simulated errors. Prior to the addition of the algorithm, data cleaning based on non-linear mixed effects models was the most effective in all datasets and had on average a minimum of 26.00% higher sensitivity and 0.12% higher specificity than other methods. Data cleaning methods using the algorithm had improved data preservation and were capable of correcting simulated errors according to the gold standard; returning a value to its original state prior to error simulation. The algorithm improved the performance of all data cleaning methods and increased the average sensitivity and specificity of the non-linear mixed effects model method by 7.68% and 0.42% respectively. Using non-linear mixed effects models combined with the algorithm to clean data allows individual growth trajectories to vary from the population by using repeated longitudinal measurements, identifies consecutive errors or those within the first data entry, avoids the requirement for a minimum number of data entries, preserves data where possible by correcting errors rather than deleting them and removes duplications intelligently. This algorithm is broadly applicable to data cleaning anthropometric data in different mammalian species and could be adapted for use in a range of other domains.

Lactulose drives a reversible reduction and qualitative modulation of the faecal microbiota diversity in healthy dogs.

Hepatic encephalopathy is a frequent and debilitating complication of liver disorders. Lactulose is an established and reasonably effective treatment, yet with incompletely understood mechanisms of action. The aims of this study were to examine how the faecal microbiota composition changed before, during and after lactulose treatment in a large animal model. Healthy, privately owned dogs (n = 18) completed a prospective cohort study. Faecal samples were collected weekly, while the subjects were either on their usual diet (week 1), or a standardised diet (weeks 2-9), with added oral lactulose in weeks 6-7. DNA extraction and 16S rRNA gene sequencing were undertaken. Faecal samples from week 7 had a significantly lower microbiota richness/diversity, based on observed operational taxonomic units, Shannon/Chao1 indexes and Pielou's Evenness. Beta diversity based on UniFrac distances was significantly different in week 7 compared to weeks 1, 5 and 9. At the phylum level, week 7 was associated with a significant increase of Firmicutes and Actinobacteria, and a decrease of Bacteroidetes and Fusobacteria, when compared to weeks 5 and 9. In summary, we have shown that lactulose induces a reversible qualitative and quantitative change of the faecal microbiota, which may explain its clinical efficacy in the management of hepatic encephalopathy.

Network analysis of canine brain morphometry links tumour risk to oestrogen deficiency and accelerated brain ageing.

Structural 'brain age' is a valuable but complex biomarker for several brain disorders. The dog is an unrivalled comparator for neurological disease modeling, however canine brain morphometric diversity creates computational and statistical challenges. Using a data-driven approach, we explored complex interactions between patient metadata, brain morphometry, and neurological disease. Twenty-four morphometric parameters measured from 286 canine brain magnetic resonance imaging scans were combined with clinical parameters to generate 9,438 data points. Network analysis was used to cluster patients according to their brain morphometry profiles. An 'aged-brain' profile, defined by a small brain width and volume combined with ventriculomegaly, was revealed in the Boxer breed. Key features of this profile were paralleled in neutered female dogs which, relative to un-neutered females, had an 11-fold greater risk of developing brain tumours. Boxer dog and geriatric dog groups were both enriched for brain tumour diagnoses, despite a lack of geriatric Boxers within the cohort. Our findings suggest that advanced brain ageing enhances brain tumour risk in dogs and may be influenced by oestrogen deficiency-a risk factor for dementia and brain tumours in humans. Morphometric features of brain ageing in dogs, like humans, might better predict neurological disease risk than patient chronological age.

Host-specialized fibrinogen-binding by a bacterial surface protein promotes biofilm formation and innate immune evasion.

Fibrinogen is an essential part of the blood coagulation cascade and a major component of the extracellular matrix in mammals. The interface between fibrinogen and bacterial pathogens is an important determinant of the outcome of infection. Here, we demonstrate that a canine host-restricted skin pathogen, Staphylococcus pseudintermedius, produces a cell wall-associated protein (SpsL) that has evolved the capacity for high strength binding to canine fibrinogen, with reduced binding to fibrinogen of other mammalian species including humans. Binding occurs via the surface-expressed N2N3 subdomains, of the SpsL A-domain, to multiple sites in the fibrinogen α-chain C-domain by a mechanism analogous to the classical dock, lock, and latch binding model. Host-specific binding is dependent on a tandem repeat region of the fibrinogen α-chain, a region highly divergent between mammals. Of note, we discovered that the tandem repeat region is also polymorphic in different canine breeds suggesting a potential influence on canine host susceptibility to S. pseudintermedius infection. Importantly, the strong host-specific fibrinogen-binding interaction of SpsL to canine fibrinogen is essential for bacterial aggregation and biofilm formation, and promotes resistance to neutrophil phagocytosis, suggesting a key role for the interaction during pathogenesis. Taken together, we have dissected a bacterial surface protein-ligand interaction resulting from the co-evolution of host and pathogen that promotes host-specific innate immune evasion and may contribute to its host-restricted ecology.

An ADAMTS3 missense variant is associated with Norwich Terrier upper airway syndrome.

In flat-faced dog breeds, air resistance caused by skull conformation is believed to be a major determinant of Brachycephalic Obstructive Airway Syndrome (BOAS). The clinical presentation of BOAS is heterogeneous, suggesting determinants independent of skull conformation contribute to airway disease. Norwich Terriers, a mesocephalic breed, are predisposed to Upper Airway Syndrome (UAS), a disease whose pathological features overlap with BOAS. Our health screening clinic examined and scored the airways of 401 Norwich terriers by laryngoscopy. Genome-wide association analyses of UAS-related pathologies revealed a genetic association on canine chromosome 13 (rs9043975, p = 7.79x10-16). Whole genome resequencing was used to identify causal variant(s) within a 414 kb critical interval. This approach highlighted an error in the CanFam3.1 dog assembly, which when resolved, led to the discovery of a c.2786G>A missense variant in exon 20 of the positional candidate gene, ADAM metallopeptidase with thrombospondin type 1 motif 3 (ADAMTS3). In addition to segregating with UAS amongst Norwich Terriers, the ADAMTS3 c.2786G>A risk allele frequency was enriched among the BOAS-susceptible French and (English) Bulldogs. Previous studies indicate that ADAMTS3 loss of function results in lymphoedema. Our results suggest a new paradigm in the understanding of canine upper airway disease aetiology: airway oedema caused by disruption of ADAMTS3 predisposes dogs to respiratory obstruction. These findings will enhance breeding practices and could refine the prognostics of surgical interventions that are often used to treat airway obstruction.

Arginine to Glutamine Variant in Olfactomedin Like 3 (OLFML3) Is a Candidate for Severe Goniodysgenesis and Glaucoma in the Border Collie Dog Breed.

Goniodysgenesis is a developmental abnormality of the anterior chamber of the eye. It is generally considered to be congenital in dogs (Canis lupus familiaris), and has been associated with glaucoma and blindness. Goniodysgenesis and early-onset glaucoma initially emerged in Border Collies in Australia in the late 1990s and have subsequently been found in this breed in Europe and the USA. The objective of the present study was to determine the genetic basis of goniodysgenesis in Border Collies. Clinical diagnosis was based on results of examinations by veterinary ophthalmologists of affected and unaffected dogs from eleven different countries. Genotyping using the Illumina high density canine single nucleotide variant genotyping chip was used to identify a candidate genetic region. There was a highly significant peak of association over chromosome 17, with a p-value of 2 × 10-13 Expression profiles and evolutionary conservation of candidate genes were assessed using public databases. Whole genome sequences of three dogs with glaucoma, three severely affected by goniodysgenesis and three unaffected dogs identified a missense variant in the olfactomedin like 3 (OLFML3) gene in all six affected animals. This was homozygous for the risk allele in all nine cases with glaucoma and 12 of 14 other severely affected animals. Of 67 reportedly unaffected animals, only one was homozygous for this variant (offspring of parents both with goniodysgenesis who were also homozygous for the variant). Analysis of pedigree information was consistent with an autosomal recessive mode of inheritance for severe goniodysgenesis (potentially leading to glaucoma) in this breed. The identification of a candidate genetic region and putative causative variant will aid breeders to reduce the frequency of goniodysgenesis and the risk of glaucoma in the Border Collie population.

Morphological variation of the caudal fossa of domestic cat skulls assessed with CT and geometric morphometrics analysis.

Objectives This study aimed to investigate differences and demonstrate a normal range of morphological variation of the caudal fossa of the cranium of domestic cats. Methods CT scans of 32 domestic cat heads of 11 breeds were included. Isosurfaces from skulls were characterised through three-dimensional geometric morphometrics using geographical landmarks placed on the internal surface of the caudal fossa and foramen magnum. Raw data was transformed with a Procrustes fit and coordinate covariance was analysed by principal components to establish breed- and sex-level differences. Skulls were also classified according to the number of concavities along the mid-sagittal vermiform impression. Differences were investigated between breed groups and sex, and correlation was sought with age. Results Analyses revealed size-independent differences in occipital bone morphology across breeds and sex; however, no clustering was evident. Most variability was observed at the exoccipital bones, ventral portion of the supraoccipital bone, dorsum sellae of the basisphenoid and the osseous tentorium cerebelli. No statistically significant differences were identified via two-sample t-tests between breed groups or sexes. No statistically significant correlation using Spearman rho correlation coefficient was identified with age. Conclusions and relevance The feline caudal fossa displays a wide range of intra- and inter-breed variation, not linked to age or sex. Concavities along the vermiform impression have not previously been described. As advanced imaging modalities are becoming more frequently used for domestic felids, an established range of normality is important for discriminating pathological changes from anatomical variances.

Canine Brachycephaly Is Associated with a Retrotransposon-Mediated Missplicing of SMOC2.

In morphological terms, "form" is used to describe an object's shape and size. In dogs, facial form is stunningly diverse. Facial retrusion, the proximodistal shortening of the snout and widening of the hard palate is common to brachycephalic dogs and is a welfare concern, as the incidence of respiratory distress and ocular trauma observed in this class of dogs is highly correlated with their skull form. Progress to identify the molecular underpinnings of facial retrusion is limited to association of a missense mutation in BMP3 among small brachycephalic dogs. Here, we used morphometrics of skull isosurfaces derived from 374 pedigree and mixed-breed dogs to dissect the genetics of skull form. Through deconvolution of facial forms, we identified quantitative trait loci that are responsible for canine facial shapes and sizes. Our novel insights include recognition that the FGF4 retrogene insertion, previously associated with appendicular chondrodysplasia, also reduces neurocranium size. Focusing on facial shape, we resolved a quantitative trait locus on canine chromosome 1 to a 188-kb critical interval that encompasses SMOC2. An intronic, transposable element within SMOC2 promotes the utilization of cryptic splice sites, causing its incorporation into transcripts, and drastically reduces SMOC2 gene expression in brachycephalic dogs. SMOC2 disruption affects the facial skeleton in a dose-dependent manner. The size effects of the associated SMOC2 haplotype are profound, accounting for 36% of facial length variation in the dogs we tested. Our data bring new focus to SMOC2 by highlighting its clinical implications in both human and veterinary medicine.

Analysis of large versus small dogs reveals three genes on the canine X chromosome associated with body weight, muscling and back fat thickness.

Domestic dog breeds display significant diversity in both body mass and skeletal size, resulting from intensive selective pressure during the formation and maintenance of modern breeds. While previous studies focused on the identification of alleles that contribute to small skeletal size, little is known about the underlying genetics controlling large size. We first performed a genome-wide association study (GWAS) using the Illumina Canine HD 170,000 single nucleotide polymorphism (SNP) array which compared 165 large-breed dogs from 19 breeds (defined as having a Standard Breed Weight (SBW) >41 kg [90 lb]) to 690 dogs from 69 small breeds (SBW ≤41 kg). We identified two loci on the canine X chromosome that were strongly associated with large body size at 82-84 megabases (Mb) and 101-104 Mb. Analyses of whole genome sequencing (WGS) data from 163 dogs revealed two indels in the Insulin Receptor Substrate 4 (IRS4) gene at 82.2 Mb and two additional mutations, one SNP and one deletion of a single codon, in Immunoglobulin Superfamily member 1 gene (IGSF1) at 102.3 Mb. IRS4 and IGSF1 are members of the GH/IGF1 and thyroid pathways whose roles include determination of body size. We also found one highly associated SNP in the 5'UTR of Acyl-CoA Synthetase Long-chain family member 4 (ACSL4) at 82.9 Mb, a gene which controls the traits of muscling and back fat thickness. We show by analysis of sequencing data from 26 wolves and 959 dogs representing 102 domestic dog breeds that skeletal size and body mass in large dog breeds are strongly associated with variants within IRS4, ACSL4 and IGSF1.

Developmental and Evolutionary Significance of the Zygomatic Bone.

The zygomatic bone is derived evolutionarily from the orbital series. In most modern mammals the zygomatic bone forms a large part of the face and usually serves as a bridge that connects the facial skeleton to the neurocranium. Our aim is to provide information on the contribution of the zygomatic bone to variation in midfacial protrusion using three samples; humans, domesticated dogs, and monkeys. In each case, variation in midface protrusion is a heritable trait produced by one of three classes of transmission: localized dysmorphology associated with single gene dysfunction, selective breeding, or long-term evolution from a common ancestor. We hypothesize that the shape of the zygomatic bone reflects its role in stabilizing the connection between facial skeleton and neurocranium and consequently, changes in facial protrusion are more strongly reflected by the maxilla and premaxilla. Our geometric morphometric analyses support our hypothesis suggesting that the shape of the zygomatic bone has less to do with facial protrusion. By morphometrically dissecting the zygomatic bone we have determined a degree of modularity among parts of the midfacial skeleton suggesting that these components have the ability to vary independently and thus can evolve differentially. From these purely morphometric data, we propose that the neural crest cells that are fated to contribute to the zygomatic bone experience developmental cues that distinguish them from the maxilla and premaxilla. The spatiotemporal and molecular identity of the cues that impart zygoma progenitors with their identity remains an open question that will require alternative data sets. Anat Rec, 299:1616-1630, 2016. © 2016 The Authors The Anatomical Record Published by Wiley Periodicals, Inc.