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

International audienceDomestic 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

Identification of genes involved in genetic diseases in human and dog : from dermatology to neurology

L'espèce canine (Canis lupus familiaris) rassemble plus de 350 races issues d'une sélection artificielle drastique menée par l'Homme au cours des derniers siècles. De ce fait, chaque race peut être considérée comme un isolat génétique développant chacune des affections génétiques de manière spontanée avec parfois de fortes fréquences. Ainsi, l'espèce canine constitue un modèle puissant pour identifier les gènes et allèles responsables de ces affections homologues à certaines maladies génétiques humaines. Mon premier projet a porté sur la recherche des causes génétiques de deux kératodermies plantaires chez le terrier irlandais et le dogue de Bordeaux. En combinant plusieurs analyses d'association (GWAS : « Genome Wide Association Study ») et des techniques de séquençage NGS (Next Generation Sequencing), un nouveau gène muté a été identifié pour le terrier irlandais. Concernant les dogues de Bordeaux, nous avons génotypé plus de 170 000 SNPs sur plus de 200 dogues. Une analyse de liaison génétique a permis d'identifier un locus de 20 Mb sur le chromosome 9 contenant un cluster de kératines. En combinant les données cliniques et génétiques, la kératine 16 s'avérait le meilleur gène candidat. Son séquençage complet a permis d'identifier une mutation complexe générant une protéine tronquée. Des analyses de PCR quantitatives ont révélé que ce gène muté était sous exprimé chez les individus atteints. Ce travail nous permet donc de proposer cette race de chien comme le premier modèle animal spontané de kératodermie focale non-épidermolytique (FNEPPK). En parallèle de ce projet, j'ai mené des recherches sur les bases moléculaires d'un syndrome d'automutilation acrale décrit dans plusieurs races. Cette neuropathie est homologue aux neuropathies héréditaires sensitives et autonomes (HSANs) chez l'Homme et se caractérise par une perte de sensibilité à la douleur au niveau des membres. A partir d'un GWAS, j'ai pu identifier un locus de 1,5 Mb chez le chien, dont l'orthologue humain n'est pas encore décrit chez des patients HSANs. Le séquençage complet de ce locus a mené à l'identification d'un variant situé dans une région cis-enhancer en amont d'un gène candidat. La mise en évidence d'une sous-expression du gène candidat indique que ce variant régulateur semblerait modifier l'activité de l'enhancer nous permettant de le proposer comme la mutation causale chez le chien. Cette découverte offre donc de nouvelles perspectives de recherche pour des patients humains atteints de neuropathies héréditaires sensitives. En combinant des approches génétiques complémentaires sur des modèles spontanés de maladies bien caractérisées chez le chien, j'ai pu participer à l'identification de trois nouveaux gènes, dont deux excellents candidats pour ces maladies homologues humaines. Le troisième représente un vrai modèle spontané de kératodermie K16, ouvrant ainsi des perspectives thérapeutiques qui bénéficieront à l'Homme et au chien.The dog species (Canis lupus familiaris) contains more than 350 distinct breeds resulting from human drastic selection during the last centuries. Each breed can then be considered as a genetic isolate, developing specific spontaneous genetic diseases with high frequencies. Thus, dogs constitute a powerful model to identify new genes and alleles involved in disorders homologous to human diseases. For my thesis, I worked on two main topics. The first one focused on the search of the genetic causes of two footpad keratodermas in the Irish terrier and the dogue de Bordeaux breeds. Concerning the Irish terrier, the work was conduced by Tosso Leeb’s team in the University of Berne, in collaboration with the Antagene Company. Using a Genome Wide Association Study (GWAS) and Next Generation Sequencing (NGS), the mutation in a new gene has been identified in footpad hyperkeratosis in this breed. For the dogue de Bordeaux project, we genotyped more than 170 000 SNPs on over 200 dogs. We then performed a genetic linkage study with a subset of 68 dogs, including 14 affected dogs. We identified a significant locus of 20 Mb on the canine chromosome 9 containing one keratin cluster. Comparing clinical, histopathological and immunochemistry data, keratin 16 appeared as an excellent candidate. The sequencing of the gene revealed a complex mutation leading to a non-functional truncated K16 protein. Quantitative RT-PCR analyses showed a strong decrease of the level of KRT16 expression in affected footpads. These results led to propose the dogue de Bordeaux footpad hyperkeratosis as the first spontaneous model of focal non-epidermolytic palmoplantar keratoderma (FNEPPK). In parallel, I studied an acral mutilation syndrome described in several hunting dog breeds. This neuropathy corresponds to human hereditary sensory and autonomic neuropathies (HSANs) and is characterized by an insensitivity to pain only in the limb. With a classical GWAS strategy, using 24 affected and 30 unaffected dogs, we identified a 1.5 Mb locus in dogs, the human orthologous locus being still unknown in human patients. Targeted sequencing of this locus revealed one single variant localized in a cis-enhancer region closed to a strong candidate gene. A lower level of expression of the candidate gene in affected dogs seems to show a functional effect of the mutation in the enhancer activity. These results led to propose this variant as the causative mutation for this canine neuropathy, and so this canine model as an opportunity to identify a new human HSAN gene. Combining complementary genetic approaches on spontaneous models of well characterized canine diseases, I did participated to the identification of three new genes, two of which being novel excellent candidates for the homologous human diseases. The third represents a true homologous model of KRT16 human keratoderma, opening the field to the development of new treatments benefiting to both humans and dogs

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

International audienceNaturally 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

A RETREG1 variant is associated with hereditary sensory and autonomic neuropathy with acral self-mutilation in purebred German Spitz

International audienceHereditary sensory and autonomic neuropathies (HSAN) represent a group of genetic diseases affecting the peripheral nervous system. In humans, at least 16 loci have been associated with the disorder but do not explain the disease origin of all patients. In dogs, similar conditions have been documented for decades in various breeds with a severe impact on life quality and are often referred to as acral mutilation syndrome (AMS). Causal variants in three genes have been identified to date, suggesting larger genetic heterogeneity in the dog population. Our aim was to explain the genetic etiology of an early‐onset HSAN/AMS in a purebred German Spitz. The affected dog showed progressive loss of pain sensation in the distal extremities, which led to intense licking, biting, and self‐mutilation of digits and paw pads. Whole‐genome sequencing identified a single candidate causal variant on chromosome 4 in the RETREG1 gene (c.656C>T, p.Pro219Leu). This missense variant was previously recognized as deleterious in a mixed breed dog family with similar clinical signs. Haplotype analyses and targeted genotyping revealed a likely German Spitz ancestry of these mixed breed dogs. Further screening of an extensive cohort of ~900 000 dogs of various breeds hinted at the variant allele origin in the German Spitz breed. Disruption of RETREG1 inhibits endoplasmic reticulum turnover and leads to neuron degeneration. Our findings provide evidence that this variant underlies the recessive form of HSAN/AMS in the German Spitz and support the use of whole‐genome sequencing‐based veterinary precision medicine for early diagnosis and prevention via a genetic test

Conservation of <i>IGSF1</i> mutated codons (in bold) between mammals.

<p>Conservation of <i>IGSF1</i> mutated codons (in bold) between mammals.</p

Derived allele frequencies at multiple loci involved in body weight and size variations in (A) modern dog breeds, (B) modern dog breeds without inclusion of muscled breeds, (C) only muscled breeds.

<p>The frequency of the derived allele in 5-kg weigh classes is represented on a color scale. Dogs with a SBW above 65 kg are collapsed in a single category (>65 kg) due to the lack of genotype variation in the group at these markers. Muscled breeds include the Boston Terrier (BOST), French Bulldog (FBUL), Miniature Bull Terrier (MBLT), Staffordshire Bull Terrier (STAF), Chinese Shar-pei (SHAR), Bulldog (BULD), Chow-Chow (CHOW), American Staffordshire Terrier (AMST), Boxer (BOX), Beauceron (BEAU), Bullmastiff (BULM), Bernese Mountain Dog (BMD), Rottweiler (ROTT), Leonberger (LEON), Tibetan Mastiff (TIBM), Neapolitan Mastiff (NEAM), Newfoundland (NEWF), Dogue de Bordeaux DDBX), English Mastiff (MAST) and Saint Bernard (STBD).</p

Observed genotypes for the ten most strongly associated variants identified in large dogs using WGS data.

<p>The three first columns correspond to the dog, breed, sex and standard breed weight (SBW). The next 10 columns correspond to the 10 most strongly associated variants at locus 1, identified from WGS data. The first part of the table corresponds to large dogs (SBW >41 kg). Homozygous and hemizygous genotypes for the “large allele” are colored in red, homozygous/hemizygous genotypes for the “small/medium” allele are colored in blue and heterozygous genotypes are colored in yellow. The second part of the table shows the distribution of the “large allele” in the 140 dogs with a SBW ≤41 kg. Values correspond to the percentage of this control population showing each genotype by variant. The last row shows the respective p-value estimated (Wald test) for each variant.</p

Best SNP markers associated with SBW above 41 kg in dogs.

<p>Best SNP markers associated with SBW above 41 kg in dogs.</p

Conservation of <i>IRS4</i> mutated codons (in bold) between mammals.

<p>Conservation of <i>IRS4</i> mutated codons (in bold) between mammals.</p