GWAS using low-pass whole genome sequence reveals a novel locus in canine congenital idiopathic megaesophagus.

Congenital idiopathic megaesophagus (CIM) is a gastrointestinal disorder of dogs wherein the esophagus is dilated and swallowing activity is reduced, causing regurgitation of ingesta. Affected individuals experience weight loss and malnourishment and are at risk for aspiration pneumonia, intussusception, and euthanasia. Great Danes have among the highest incidences of CIM across dog breeds, suggesting a genetic predisposition. We generated low-pass sequencing data for 83 Great Danes and used variant calls to impute missing whole genome single-nucleotide variants (SNVs) for each individual based on haplotypes phased from 624 high-coverage dog genomes, including 21 Great Danes. We validated the utility of our imputed data set for genome-wide association studies (GWASs) by mapping loci known to underlie coat phenotypes with simple and complex inheritance patterns. We conducted a GWAS for CIM with 2,010,300 SNVs, identifying a novel locus on canine chromosome 1 (P-val = 2.76 × 10-10). Associated SNVs are intergenic or intronic and are found in two clusters across a 1.7-Mb region. Inspection of coding regions in high-coverage genomes from affected Great Danes did not reveal candidate causal variants, suggesting that regulatory variants underlie CIM. Further studies are necessary to assess the role of these non-coding variants.

Congenital idiopathic megaesophagus in the German shepherd dog is a sex-differentiated trait and is associated with an intronic variable number tandem repeat in Melanin-Concentrating Hormone Receptor 2.

Congenital idiopathic megaesophagus (CIM) is a gastrointestinal (GI) motility disorder of dogs in which reduced peristaltic activity and dilation of the esophagus prevent the normal transport of food into the stomach. Affected puppies regurgitate meals and water, fail to thrive, and experience complications such as aspiration pneumonia that may necessitate euthanasia. The German shepherd dog (GSD) has the highest disease incidence, indicative of a genetic predisposition. Here, we discover that male GSDs are twice as likely to be affected as females and show that the sex bias is independent of body size. We propose that female endogenous factors (e.g., estrogen) are protective via their role in promoting relaxation of the sphincter between the esophagus and stomach, facilitating food passage. A genome-wide association study for CIM revealed an association on canine chromosome 12 (P-val = 3.12x10-13), with the lead SNPs located upstream or within Melanin-Concentrating Hormone Receptor 2 (MCHR2), a compelling positional candidate gene having a role in appetite, weight, and GI motility. Within the first intron of MCHR2, we identified a 33 bp variable number tandem repeat (VNTR) containing a consensus binding sequence for the T-box family of transcription factors. Across dogs and wolves, the major allele includes two copies of the repeat, whereas the predominant alleles in GSDs have one or three copies. The single-copy allele is strongly associated with CIM (P-val = 1.32x10-17), with homozygosity for this allele posing the most significant risk. Our findings suggest that the number of T-box protein binding motifs may correlate with MCHR2 expression and that an imbalance of melanin-concentrating hormone plays a role in CIM. We describe herein the first genetic factors identified in CIM: sex and a major locus on chromosome 12, which together predict disease state in the GSD with greater than 75% accuracy.

Multi-omics approach identifies germline regulatory variants associated with hematopoietic malignancies in retriever dog breeds.

Histiocytic sarcoma is an aggressive hematopoietic malignancy of mature tissue histiocytes with a poorly understood etiology in humans. A histologically and clinically similar counterpart affects flat-coated retrievers (FCRs) at unusually high frequency, with 20% developing the lethal disease. The similar clinical presentation combined with the closed population structure of dogs, leading to high genetic homogeneity, makes dogs an excellent model for genetic studies of cancer susceptibility. To determine the genetic risk factors underlying histiocytic sarcoma in FCRs, we conducted multiple genome-wide association studies (GWASs), identifying two loci that confer significant risk on canine chromosomes (CFA) 5 (Pwald = 4.83x10-9) and 19 (Pwald = 2.25x10-7). We subsequently undertook a multi-omics approach that has been largely unexplored in the canine model to interrogate these regions, generating whole genome, transcriptome, and chromatin immunoprecipitation sequencing. These data highlight the PI3K pathway gene PIK3R6 on CFA5, and proximal candidate regulatory variants that are strongly associated with histiocytic sarcoma and predicted to impact transcription factor binding. The CFA5 association colocalizes with susceptibility loci for two hematopoietic malignancies, hemangiosarcoma and B-cell lymphoma, in the closely related golden retriever breed, revealing the risk contribution this single locus makes to multiple hematological cancers. By comparison, the CFA19 locus is unique to the FCR and harbors risk alleles associated with upregulation of TNFAIP6, which itself affects cell migration and metastasis. Together, these loci explain ~35% of disease risk, an exceptionally high value that demonstrates the advantages of domestic dogs for complex trait mapping and genetic studies of cancer susceptibility.

Genetic analysis of the modern Australian labradoodle dog breed reveals an excess of the poodle genome.

The genomic diversity of the domestic dog is an invaluable resource for advancing understanding of mammalian biology, evolutionary biology, morphologic variation, and behavior. There are approximately 350 recognized breeds in the world today, many established through hybridization and selection followed by intense breeding programs aimed at retaining or enhancing specific traits. As a result, many breeds suffer from an excess of particular diseases, one of many factors leading to the recent trend of "designer breed" development, i.e. crossing purebred dogs from existing breeds in the hope that offspring will be enriched for desired traits and characteristics of the parental breeds. We used a dense panel of 150,106 SNPs to analyze the population structure of the Australian labradoodle (ALBD), to understand how such breeds are developed. Haplotype and admixture analyses show that breeds other than the poodle (POOD) and Labrador retriever (LAB) contributed to ALBD formation, but that the breed is, at the genetic level, predominantly POOD, with all small and large varieties contributing to its construction. Allele frequency analysis reveals that the breed is enhanced for variants associated with a poodle-like coat, which is perceived by breeders to have an association with hypoallergenicity. We observed little enhancement for LAB-specific alleles. This study provides a blueprint for understanding how dog breeds are formed, highlighting the limited scope of desired traits in defining new breeds.

Variants in FtsJ RNA 2'-O-Methyltransferase 3 and Growth Hormone 1 are associated with small body size and a dental anomaly in dogs.

Domesticated dogs show unparalleled diversity in body size across breeds, but within breeds variation is limited by selective breeding. Many heritable diseases of dogs are found among breeds of similar sizes, suggesting that as in humans, alleles governing growth have pleiotropic effects. Here, we conducted independent genome-wide association studies in the small Shetland Sheepdog breed and discovered a locus on chromosome 9 that is associated with a dental abnormality called maxillary canine-tooth mesioversion (MCM) (P = 1.53 × 10-7) as well as two body size traits: height (P = 1.67 × 10-5) and weight (P = 1.16 × 10-7). Using whole-genome resequencing data, we identified variants in two proximal genes: FTSJ3, encoding an RNA methyltransferase, and GH1, encoding growth hormone. A substitution in FTSJ3 and a splice donor insertion in GH1 are strongly associated with MCM and reduced body size in Shetland Sheepdogs. We demonstrated in vitro that the GH1 variant leads to exon 3 skipping, predicting a mutant protein known to cause human pituitary dwarfism. Statistical modeling, however, indicates that the FTSJ3 variant is the stronger predictor of MCM and that each derived allele reduces body size by about 1 inch and 5 pounds. In a survey of 224 breeds, both FTSJ3 and GH1 variants are frequent among very small "toy" breeds and absent from larger breeds. Our findings indicate that a chromosome 9 locus harboring tightly linked variants in FTSJ3 and GH1 reduces growth in the Shetland Sheepdog and toy breed dogs and confers risk for MCM through vertical pleiotropy.

Congenital myasthenic syndrome in Golden Retrievers is associated with a novel COLQ mutation.

BACKGROUND: Congenital myasthenic syndromes (CMSs) are a group of inherited disorders of neuromuscular transmission that may be presynaptic, synaptic, or postsynaptic. Causative mutations have been identified in 4 breeds including the Labrador Retriever, Jack Russell Terrier, Heideterrier, and Danish Pointing Dog. HYPOTHESIS/OBJECTIVE: Clinical and genetic characterization of a neuromuscular disorder in Golden Retriever (GR) puppies. ANIMALS: Four GR puppies from California were evaluated for generalized muscle weakness beginning at weaning. Biological specimens were collected from the affected puppies, and familial information was obtained. Blood or buccal swabs were obtained from 63 unaffected GRs. METHODS: Complete physical, neurological, electrodiagnostic, and histological evaluations and biochemical quantification of muscle acetylcholine receptors were performed. Polymerase chain reaction was used to amplify the 17 exons of COLQ, and sequences were obtained by Sanger sequencing. Variant frequency was assessed in unrelated GRs and a public database. RESULTS: Clinical, neurological, and electrodiagnostic evaluations confirmed a disorder of neuromuscular transmission in a GR family. Sequencing of all exons and splice sites of a primary candidate gene, COLQ, identified a point mutation that predicts an amino acid substitution (G294R). The primary COLQ transcript was absent from affected muscle samples. All affected puppies were homozygous for the mutation, which was not detected outside this GR family or in other breeds. CONCLUSIONS AND CLINICAL IMPORTANCE: We confirmed the diagnosis of a CMS in GR puppies and identified a novel COLQ mutation. The COLQ gene encodes the collagenous tail of acetylcholinesterase, the enzyme responsible for termination of skeletal muscle contraction by clearing acetylcholine at the neuromuscular junction. Clinicians and breeders should be aware of this CMS in GR puppies with an early onset of weakness.

Novel Y Chromosome Retrocopies in Canids Revealed through a Genome-Wide Association Study for Sex.

The lack of an annotated reference sequence for the canine Y chromosome has limited evolutionary studies, as well as our understanding of the role of Y-linked sequences in phenotypes with a sex bias. In genome-wide association studies (GWASs), we observed spurious associations with autosomal SNPs when sex was unbalanced in case-control cohorts and hypothesized that a subset of SNPs mapped to autosomes are in fact sex-linked. Using the Illumina 230K CanineHD array in a GWAS for sex, we identified SNPs that amplify in both sexes but possess significant allele frequency differences between males and females. We found 48 SNPs mapping to 14 regions of eight autosomes and the X chromosome that are Y-linked, appearing heterozygous in males and monomorphic in females. Within these 14 regions are eight genes: three autosomal and five X-linked. We investigated the autosomal genes (MITF, PPP2CB, and WNK1) and determined that the SNPs are diverged nucleotides in retrocopies that have transposed to the Y chromosome. MITFY and WNK1Y are expressed and appeared recently in the Canidae lineage, whereas PPP2CBY represents a much older insertion with no evidence of expression in the dog. This work reveals novel canid Y chromosome sequences and provides evidence for gene transposition to the Y from autosomes and the X.

Canine Cancer Genomics: Lessons for Canine and Human Health.

Dogs are second only to humans in medical surveillance and preventative health care, leading to a recent perception of increased cancer incidence. Scientific priorities in veterinary oncology have thus shifted, with a demand for cancer genetic screens, better diagnostics, and more effective therapies. Most dog breeds came into existence within the last 300 years, and many are derived from small numbers of founders. Each has undergone strong artificial selection, in which dog fanciers selected for many traits, including body size, fur type, color, skull shape, and behavior, to create novel breeds. The adoption of the breed barrier rule-no dog may become a registered member of a breed unless both its dam and its sire are registered members-ensures a relatively closed genetic pool within each breed. As a result, there is strong phenotypic homogeneity within breeds but extraordinary phenotypic variation between breeds. One consequence of this is the high level of breed-associated genetic disease. We and others have taken advantage of this to identify genes for a large number of canine maladies for which mouse models do not exist, particularly with regard to cancer.

Length variations within the Merle retrotransposon of canine PMEL: correlating genotype with phenotype.

BACKGROUND: The antisense insertion of a canine short interspersed element (SINEC_Cf) in the pigmentation gene PMEL (or SILV) causes a coat pattern phenotype in dogs termed merle. Merle is a semi-dominant trait characterized by patches of full pigmentation on a diluted background. The oligo(dT) tract of the Merle retrotransposon is long and uninterrupted and is prone to dramatic truncation. Phenotypically wild-type individuals carrying shorter oligo(dT) lengths of the Merle allele have been previously described and termed cryptic merles. Two additional coat patterns, dilute merle (uniform, steely-grey coat) and harlequin merle (white background with black patches), also appear in breeds segregating the Merle allele. RESULTS: Sequencing of all PMEL exons in a dilute and a harlequin merle reveals that variation exists solely within the oligo(dT) tract of the SINEC_Cf insertion. In fragment analyses from 259 dogs heterozygous for Merle, we observed a spectrum of oligo(dT) lengths spanning 25 to 105 base pairs (bp), with ranges that correspond to the four varieties of the merle phenotype: cryptic (25-55 bp), dilute (66-74 bp), standard (78-86 bp), and harlequin (81-105 bp). Somatic contractions of the oligo(dT) were observed in 43% of standard and 51% of harlequin merle dogs. A small proportion (4.6%) of the study cohort inherited de novo contractions or expansions of the Merle allele that resulted in dilute or harlequin coat patterns, respectively. CONCLUSIONS: The phenotypic consequence of the Merle SINE insertion directly depends upon oligo(dT) length. In transcription, we propose that the use of an alternative splice site increases with oligo(dT) length, resulting in insufficient PMEL and a pigment dilution spectrum, from dark grey to complete hypopigmentation. We further propose that during replication, contractions and expansions increase in frequency with oligo(dT) length, causing coat variegation (somatic events in melanocytes) and the spontaneous appearance of varieties of the merle phenotype (germline events).

Exome sequencing reveals independent SGCD deletions causing limb girdle muscular dystrophy in Boston terriers.

BACKGROUND: Limb-girdle muscular dystrophies (LGMDs) are a heterogeneous group of inherited autosomal myopathies that preferentially affect voluntary muscles of the shoulders and hips. LGMD has been clinically described in several breeds of dogs, but the responsible mutations are unknown. The clinical presentation in dogs is characterized by marked muscle weakness and atrophy in the shoulder and hips during puppyhood. METHODS: Following clinical evaluation, the identification of the dystrophic histological phenotype on muscle histology, and demonstration of the absence of sarcoglycan-sarcospan complex by immunostaining, whole exome sequencing was performed on five Boston terriers: one affected dog and its three family members and one unrelated affected dog. RESULTS: Within sarcoglycan-δ (SGCD), a two base pair deletion segregating with LGMD in the family was discovered, and a deletion encompassing exons 7 and 8 was found in the unrelated dog. Both mutations are predicted to cause an absence of SGCD protein, confirmed by immunohistochemistry. The mutations are private to each family. CONCLUSIONS: Here, we describe the first cases of canine LGMD characterized at the molecular level with the classification of LGMD2F.

Beyond the MHC: A canine model of dermatomyositis shows a complex pattern of genetic risk involving novel loci.

Juvenile dermatomyositis (JDM) is a chronic inflammatory myopathy and vasculopathy driven by genetic and environmental influences. Here, we investigated the genetic underpinnings of an analogous, spontaneous disease of dogs also termed dermatomyositis (DMS). As in JDM, we observed a significant association with a haplotype of the major histocompatibility complex (MHC) (DLA-DRB1*002:01/-DQA1*009:01/-DQB1*001:01), particularly in homozygosity (P-val = 0.0001). However, the high incidence of the haplotype among healthy dogs indicated that additional genetic risk factors are likely involved in disease progression. We conducted genome-wide association studies in two modern breeds having common ancestry and detected strong associations with novel loci on canine chromosomes 10 (P-val = 2.3X10-12) and 31 (P-val = 3.95X10-8). Through whole genome resequencing, we identified primary candidate polymorphisms in conserved regions of PAN2 (encoding p.Arg492Cys) and MAP3K7CL (c.383_392ACTCCACAAA>GACT) on chromosomes 10 and 31, respectively. Analyses of these polymorphisms and the MHC haplotypes revealed that nine of 27 genotypic combinations confer high or moderate probability of disease and explain 93% of cases studied. The pattern of disease risk across PAN2 and MAP3K7CL genotypes provided clear evidence for a significant epistatic foundation for this disease, a risk further impacted by MHC haplotypes. We also observed a genotype-phenotype correlation wherein an earlier age of onset is correlated with an increased number of risk alleles at PAN2 and MAP3K7CL. High frequencies of multiple genetic risk factors are unique to affected breeds and likely arose coincident with artificial selection for desirable phenotypes. Described herein is the first three-locus association with a complex canine disease and two novel loci that provide targets for exploration in JDM and related immunological dysfunction.

Exome sequencing reveals a nebulin nonsense mutation in a dog model of nemaline myopathy.

Nemaline myopathy (NM) is a congenital muscle disorder associated with muscle weakness, hypotonia, and rod bodies in the skeletal muscle fibers. Mutations in 10 genes have been implicated in human NM, but spontaneous cases in dogs have not been genetically characterized. We identified a novel recessive myopathy in a family of line-bred American bulldogs (ABDs); rod bodies in muscle biopsies established this as NM. Using SNP profiles from the nuclear family, we evaluated inheritance patterns at candidate loci and prioritized TNNT1 and NEB for further investigation. Whole exome sequencing of the dam, two affected littermates, and an unaffected littermate revealed a nonsense mutation in NEB (g.52734272 C>A, S8042X). Whole tissue gel electrophoresis and western blots confirmed a lack of full-length NEB in affected tissues, suggesting nonsense-mediated decay. The pathogenic variant was absent from 120 dogs of 24 other breeds and 100 unrelated ABDs, suggesting that it occurred recently and may be private to the family. This study presents the first molecularly characterized large animal model of NM, which could provide new opportunities for therapeutic approaches.