Guillaume Queney

Progressive retinal atrophy in the Border Collie: a new XLPRA.

BackgroundSeveral forms of progressive retinal atrophy (PRA) segregate in more than 100 breeds of dog with each PRA segregating in one or a few breeds. This breed specificity may be accounted for by founder effects and genetic drift, which have reduced the genetic heterogeneity of each breed, thereby facilitating the identification of causal mutations. We report here a new form of PRA segregating in the Border Collie breed. The clinical signs, including the loss of night vision and a progressive loss of day vision, resulting in complete blindness, occur at the age of three to four years and may be detected earlier through systematic ocular fundus examination and electroretinography (ERG).ResultsOphthalmic examinations performed on 487 dogs showed that affected dogs present a classical form of PRA. Of those, 274 have been sampled for DNA extraction and 87 could be connected through a large pedigree. Segregation analysis suggested an X-linked mode of transmission; therefore both XLPRA1 and XLPRA2 mutations were excluded through the genetic tests.ConclusionHaving excluded these mutations, we suggest that this PRA segregating in Border Collie is a new XLPRA (XLPRA3) and propose it as a potential model for the homologous human disease, X-Linked Retinitis Pigmentosa.

A Spontaneous KRT16 Mutation in a Dog Breed: A Model for Human Focal Non-Epidermolytic Palmoplantar Keratoderma (FNEPPK)

TO THE EDITOR The keratin 16 gene (KRT16) encodes an intermediate filament protein mainly expressed in palmoplantar epidermis. In humans, mutations in KRT16 are responsible for pachyonychia congenita and focal non-epidermolytic palmoplantar keratoderma (FNEPPK; Smith et al., 2000; McLean and Moore, 2011). One of the main symptoms is a painful thickening of the palms and soles. To understand molecular mechanisms involved in this keratoderma, Krt16 mutant mouse models have been developed, but only one reproduces fully the palmoplantar phenotype (Lessard and Coulombe, 2012). In this study, we present a spontaneous canine model of FNEPPK inherited as an autosomal recessive disorder in the Dogue de Bordeaux breed. Because of its population structure, which features genetic isolates, the purebred dog model has recently proven its utility in understanding the molecular mechanisms of hereditary cornification disorders, notably in humans and dog Autosomal Recessive Congenital Ichthyosis (Grall et al., 2012). We investigated a family of 130 dogs including 28 affected animals; no sex bias was observed among the 13 males and 15 females analyzed. The onset usually occurred between 10 weeks and 1 year of age. First described by Paradis (1992), affected dogs exhibit a painful thickening of the footpads with severe keratinous proliferations and fissures only at the ground contact locations similar to those observed in FNEPPK patients (Figure 1). Cracks predispose the dogs to secondary infections, leading to lameness, causing the dog to be reluctant to walk. Nails did not seem to be affected, as reported in some human FNEPPK patients and in Krt16-null mice models (Shamsher et al., 1995; Smith et al., 2000; Liao et al., 2007; Lessard and Coulombe, 2012). Similarly, no other cutaneous sign such as oral leukoplakia, cysts, or follicular keratosis was reported. This is concordant with our results of quantitative reverse transcription PCR of messsengerRNA from unaffected dog biopsies, showing strong and specific expression of KRT16 in the footpad, nose, and keratinocytes but not in body skin, oral mucosa, or other organs (data not shown). Histopathological examinations of footpad biopsies revealed thick hyperkeratotic digital epidermis that was roughened by marked conical papillae with a prominent ‘‘church spire’’ appearance (Figure 1e). On the top of this, there is a thin compact column of parakeratotic cells with absent or decreased underlying granular layer and cytoplasmic vacuolization of superficial corneocytes at their base (Figure 1f and g). Outside the conical papillae, the epidermis exhibited a well-developed granular layer and compact orthohyperkeratosis. Between the conical papillae, small valleys were observed that presented dyskeratosis, an irregular and prominent granular layer, and light to moderate keratotic plugging (Figure 1h). No epidermolytic changes were noticed. Chronic superficial perivascular dermal infiltrate was sometimes observed. Immunohistochemistry and immunofluorescence staining were performed on FFPE skin biopsies from four affected and four unaffected dogs. The Ki67 proliferation index showed that, as expected, keratinocytes in affected footpads were not proliferating. Expression of keratin 1, 6, and 16 was investigated in normal footpad biopsies (Figure 2). As previously described (Bowden et al., 2009), keratin 1, 6, and 16 are coexpressed in the suprabasal layer of the footpad epidermis, with K16 located in the center of the rete ridges. No differences were observed in the expression of K1 and K6 between cases and controls. However, immunostaining revealed an abnormal distribution of K16 in affected samples: although K16 expression was diffuse and suprabasal all over the thickness of the epithelial layer of control dogs, its expression was not detected in affected samples (Figure 2). In affected dogs, discrete aggregates of K16 could be observed in the cornified layer in footpads not in contact with the ground (Supplementary Figure S1a online). In parallel, we performed a genetic linkage study on the Dogue de Bordeaux family using 14 affected and 54 unaffected dogs genotyped on the canine 173,000 SNP array Q3 . We identified a 20 Mb locus corresponding to the dog type I keratin cluster. We carried out mutation screening on several keratins in 14 affected dogs and 16 controls and identified a complex mutation in KRT16 corresponding to an insertion/ deletion (indel) of four nucleotides and a separate 1 bp deletion 15 nucleotides downstream in exon 6 (Supplementary Figure S2 online). This complex indel results in an insertion of 1 bp in affected dogs and introduces a frameshift changing the sequence of 10 amino acids and creating a premature stop codon (p.Glu392*) in the open reading frame of the gene. This stop codon located in the 2B domain leads to the loss of the last 85 amino acids of K16, including the helix termination motif LETTER TO THE EDITOR

Female in the inside, male in the outside: insights into the spatial organization of a European wildcat population

Hybridization between the European wildcat, Felissilvestris silvestris, and the domestic cat, Felissilvestris catus, has been found in several European countries with different landscape structures and in various proportions. In this study, we focus on a local population of European wildcats in forests fragmented by agricultural lands in northeastern France. Our aim is to better understand how the spatial organization of the wildcats in this particular type of environment might impact the proportion of hybridization. We combined radio-tracking and genetics through the use of microsatellite markers in order to assess both the spacing pattern and the level of hybridization of this wildcat population. Hybridization is rare in this wildcat population with only one putative hybrid (most likely backcrossed) detected out of 42 putative wildcats. We found that most females were concentrated inside the forest while males stood in the periphery or outside the forest. Furthermore, many males and females resulted related. Such a spacing pattern might limit contacts between male domestic cats and female wildcats and can be one of the causes that explain the low level of hybridization in the wildcat population in this environment. We could not exclude the possibility of hybrid presence in the neighboring domestic cat populations. Our results yield new insights on the influence that the landscape configuration and the spacing pattern can have on genetic flow between the populations of the two subspecies.

Contrasted hybridization patterns between two local populations of European wildcats in France

The European wildcat (Felis silvestris silvestris) is threatened across the totality of its area of distribution by hybridization with the domestic cat F.s. catus. The underlying ecological processes promoting hybridization, remain largely unknown. In France, wildcats are mainly present in the North-East but signs of their presence in the Pyrenees have been recently provided. However, no studies have been carried out in the French Pyrenees to assess the genetic status of wildcats. We have compared a local population of wildcats living in a continuous habitat in the French Pyrenees and a local population of wildcats living in a fragmented habitat in Northeastern France to evaluate how habitat fragmentation influence the population structure of European wildcats. Close kin were not found in the same geographic location contrary to what was observed for females in the Northeastern wildcat population. Furthermore, there was no evidence of hybridization in the Pyrenean wildcats and only one domestic cat raised suspicions while hybridization was categorically detected in northeastern France. The two wildcat populations were significantly differentiated (Fst = 0.08) and the genetic diversity of the Pyrenean wildcats was lower than that of other wildcat populations in France and in Europe. Taken together, these results suggest that habitat fragmentation, and in particular the absence of agricultural fields, may play an important role in lowering the probability of hybridization by reducing the likelihood of contact with domestic cats. Moreover, our results suggest that the French Pyrenean wildcat populations is isolated and may be threatened by a lack of genetic diversity.The European wildcat (Felis silvestris silvestris) is threatened across the totality of its area of distribution by hybridization with the domestic cat F.s. catus. The underlying ecological processes promoting hybridization, remain largely unknown. In France, wildcats are mainly present in the North-East but signs of their presence in the Pyrenees have been recently provided. However, no studies have been carried out in the French Pyrenees to assess the genetic status of wildcats. We have compared a local population of wildcats living in a continuous habitat in the French Pyrenees and a local population of wildcats living in a fragmented habitat in Northeastern France to evaluate how habitat fragmentation influence the population structure of European wildcats. Close kin were not found in the same geographic location contrary to what was observed for females in the Northeastern wildcat population. Furthermore, there was no evidence of hybridization in the Pyrenean wildcats and only one domestic cat raised suspicions while hybridization was categorically detected in northeastern France. The two wildcat populations were significantly differentiated (Fst = 0.08) and the genetic diversity of the Pyrenean wildcats was lower than that of other wildcat populations in France and in Europe. Taken together, these results suggest that habitat fragmentation, and in particular the absence of agricultural fields, may play an important role in lowering the probability of hybridization by reducing the likelihood of contact with domestic cats. Moreover, our results suggest that the French Pyrenean wildcat populations is isolated and may be threatened by a lack of genetic diversity.

A set of 20 multiplexed single nucleotide polymorphism (SNP) markers specifically selected for the identification of the wild boar (Sus scrofa scrofa) and the domestic pig (Sus scrofa domesticus)

In the last few decades, populations of wild boars have been spreading throughout Europe. This expansion is problematic because of conflicts with human activities and/or sanitary reasons. Thus, the need to develop tools to understand wild boar populations’ increase as well as to manage efficiently their populations appears crucial but challenging. As ancestor of the domestic pig, one of the main issues associated with this population spread is the possible hybridization between the two subspecies which complicates the identification of pure individuals. In this study, we propose a set of 20 single nucleotide polymorphism (SNP) markers, allocated in two 10-plexes, selected in order to identify the two subspecies. The multiplexes were validated on reference populations of wild boars and domestic pigs in order to assess their polymorphism and their discriminative power. The selected markers allowed for an exacerbated differentiation between the wild boars and the domestic pigs compared to other studies and thus allows for an efficient identification of species. The low size of this panel of 20 SNP markers makes it a tool compatible with economic constraints associated with wildlife management compared to tools based on chips.

Les tests génétiques chez le chien

Resume Avec pres de 400 races, le chien est l’espece de mammifere qui possede la plus grande diversite morphologique, comportementale et de sensibilite aux maladies genetiques. Cette diversite tient a l’histoire evolutive du chien et a la creation d’autant de races distinctes par l’homme depuis plusieurs siecles. L’avenement des techniques de biologie moleculaire et le developpement d’outils genetiques performants ont permis au chien d’entrer dans l’ere de la genetique moleculaire. Depuis une petite dizaine d’annees, les veterinaires praticiens disposent de tests ADN pour le diagnostic et le depistage de certaines maladies hereditaires canines invalidantes. Ces outils diagnostiques et predictifs complementaires leur permettent de conseiller les proprietaires et eleveurs canins. Cependant, seul un nombre restreint de tests est commercialise en France et les multiples possibilites qu’offre leur usage ne doit pas faire oublier les precautions elementaires d’emploi de ces tests. Apres quelques rappels concernant la genetique canine, cet article s’interesse aux tests ADN disponibles en France pour le chien et aux etapes qui ont conduit de la caracterisation clinique d’une nouvelle maladie hereditaire a la validation et la commercialisation d’un test. L’etat de la recherche en France dans ce domaine est illustre par les projets de genetique canine en cours et leur interet en pathologie comparee, ainsi que par la constitution d’une bio-banque de prelevements de chiens (CaniDNA) pour l’etude des maladies genetiques. Les maladies hereditaires oculaires, particulierement nombreuses et bien etudiees chez le chien, font l’objet d’un paragraphe specifique. Enfin, les applications en genetique feline sont evoquees.