A Spontaneous KRT16 Mutation in a Dog Breed:a Model for Human Focal Non-Epidermolytic PalmoplantarKeratoderma (FNEPPK)Journal of Investigative Dermatology accepted article preview 18 December 2014; doi:10.1038/jid.2014.526

TO THE EDITORThe keratin 16 gene (KRT16) encodes anintermediate filament protein mainlyexpressed in palmoplantar epidermis.In humans, mutations in KRT16 areresponsible for pachyonychia congenitaand focal non-epidermolytic palmoplan-tar keratoderma (FNEPPK; Smith et al.,2000; McLean and Moore, 2011). Oneof the main symptoms is a painfulthickening of the palms and soles. Tounderstand molecular mechanismsinvolved in this keratoderma, Krt16 mu-tant mouse models have been develo-ped, but only one reproduces fully thepalmoplantar phenotype (Lessard andCoulombe, 2012). In this study, wepresent a spontaneous canine model ofFNEPPK inherited as an autosomal re-cessive disorder in the Dogue de Borde-aux breed. Because of its populationstructure, which features geneticisolates, the purebred dog model hasrecently proven its utility in understand-ing the molecular mechanisms ofhereditary cornification disorders, nota-bly in humans and dog AutosomalRecessive Congenital Ichthyosis (Grallet al., 2012).

We investigated a family of 130 dogsincluding 28 affected animals; no sexbias was observed among the 13 malesand 15 females analyzed. The onsetusually occurred between 10 weeksand 1 year of age. First described byParadis (1992), affected dogs exhibit apainful thickening of the footpads withsevere keratinous proliferations andfissures only at the ground contactlocations similar to those observed inFNEPPK patients (Figure 1). Cracks pre-dispose the dogs to secondary infec-tions, leading to lameness, causing thedog to be reluctant to walk. Nails didnot seem to be affected, as reported in

some human FNEPPK patients and inKrt16-null mice models (Shamsheret al., 1995; Smith et al., 2000; Liaoet al., 2007; Lessard and Coulombe,2012). Similarly, no other cutaneoussign such as oral leukoplakia, cysts, orfollicular keratosis was reported. This isconcordant with our results ofquantitative reverse transcription PCRof messsengerRNA from unaffected dogbiopsies, showing strong and specificexpression of KRT16 in the footpad,nose, and keratinocytes but not inbody skin, oral mucosa, or otherorgans (data not shown).

Histopathological examinations offootpad biopsies revealed thick hyper-keratotic digital epidermis that wasroughened by marked conical papillaewith a prominent ‘‘church spire’’ appea-rance (Figure 1e). On the top of this,there is a thin compact column ofparakeratotic cells with absent ordecreased underlying granular layerand cytoplasmic vacuolization of super-ficial corneocytes at their base (Figure 1fand g). Outside the conical papillae, theepidermis exhibited a well-developedgranular layer and compact orthohyper-keratosis. Between the conical papillae,small valleys were observed thatpresented dyskeratosis, an irregular andprominent granular layer, and light tomoderate keratotic plugging (Figure 1h).No epidermolytic changes werenoticed. Chronic superficial perivasculardermal infiltrate was sometimesobserved.

Immunohistochemistry and immuno-fluorescence staining were performedon FFPE skin biopsies from four affectedand four unaffected dogs. The Ki67proliferation index showed that, asexpected, keratinocytes in affected foot-pads were not proliferating. Expression

of keratin 1, 6, and 16 was investigatedin normal footpad biopsies (Figure 2). Aspreviously described (Bowden et al.,2009), keratin 1, 6, and 16 are co-expressed in the suprabasal layer of thefootpad epidermis, with K16 located inthe center of the rete ridges. No diffe-rences were observed in the expressionof K1 and K6 between cases andcontrols. However, immunostainingrevealed an abnormal distribution ofK16 in affected samples: although K16expression was diffuse and suprabasalall over the thickness of the epitheliallayer of control dogs, its expression wasnot detected in affected samples(Figure 2). In affected dogs, discreteaggregates of K16 could be observedin the cornified layer in footpads not incontact with the ground (SupplementaryFigure S1a online).

In parallel, we performed a geneticlinkage study on the Dogue de Bor-deaux family using 14 affected and 54unaffected dogs genotyped on thecanine 173,000 SNP array

Q3. We identi-

fied a 20 Mb locus corresponding to thedog type I keratin cluster. We carriedout mutation screening on several kera-tins in 14 affected dogs and 16 controlsand identified a complex mutation inKRT16 corresponding to an insertion/deletion (indel) of four nucleotides and aseparate 1 bp deletion 15 nucleotidesdownstream in exon 6 (SupplementaryFigure S2 online). This complex indelresults in an insertion of 1 bp in affecteddogs and introduces a frameshift chan-ging the sequence of 10 amino acidsand creating a premature stop codon(p.Glu392*) in the open reading frameof the gene. This stop codon locatedin the 2B domain leads to the lossof the last 85 amino acids of K16,including the helix termination motif

LETTER TO THE EDITOR

& 2014 The Society for Investigative Dermatology www.jidonline.org 1

(Supplementary Figure S2 online). Thissequence is the most highly conservedmotif in keratins involved in the end-to-

end interactions in keratin assembly.Mutations removing or changing thesequence of the tail domain leading to

the production of truncated or mutatedkeratins have already been described forkeratin 10 in humans and dog patients

Figure 1. Clinical phenotype and histopathological findings of footpad keratoderma in the Dogue de Bordeaux. (a) Footpad of a 3-year-old unaffected dog. (b)

Footpad of an 8-year old and (c) a 1-year old affected dog. (d–h) Hematoxylin and eosin (HE) staining of footpad biopsies. (d) Unaffected dog (scale bar¼ 100mm).

(e–h) Footpad of an 8-year-old affected dog. (e) Thick hyperkeratotic digital epidermis (scale bar¼ 100mm); (f) Thin compact column of parakeratotic cells (scale

bar¼ 100mm); (g) Higher magnification reveals a thinner or even absent granular layer (scale bar¼50mm). (h) Irregular prominent granular layer, dyskeratosis, and

light keratotic plugging at the bottom of two epidermal valleys (scale bar¼100mm).

Unaffecteddog

K1 K6 K16

Affecteddog

Affecteddog

Unaffecteddog

100 µm

100 µm 100 µm

100 µm100 µm

100 µm

Figure 2. Immunoperoxidase and Immunofluorescence staining of unaffected and affected footpad biopsies with Þ¥-K1, Þ¥-K6, and Þ¥-K16. Keratin 1 (a, d, g, j)

and keratin 6 (b, e, h, k) have an expected diffuse and suprabasal expression in the epidermis in both in unaffected and affected dogs. (c, i) For keratin 16, a diffuse

and suprabasal expression is observed in the epidermis in unaffected dogs (f, l), whereas keratin 16 is weakly or not detected in the epidermis of affected dogs.

scale bar¼100mm.

J Plassais et al.A Spontaneous KRT16 Mutation in a Dog Breed

2 Journal of Investigative Dermatology (2014), Volume 00

(Credille et al., 2005; Muller et al.,2006; Gutierrez et al., 2013) and forKRT16 in human patients (Smith et al.,2000). It has been suggested that thedisruption of the filament assemblycould potentially lead to an abnormaldistribution of the protein and thecreation of aggregates (Smith et al.,2000; Muller et al., 2006) similar towhat we observed in Dogues deBordeaux (Supplementary Figure S1aonline).

To confirm that this mutation iscausative and specific to the Dogue deBordeaux, we sequenced a set of 334Dogues de Bordeaux with known clin-ical status. All affected dogs were homo-zygous for the complex mutation, andall unaffected dogs were either homo-zygous for the wild-type alleles or het-erozygous (245/306 and 61/306,respectively). Furthermore, the mutatedallele was never detected in a panel of344 unaffected dogs from 80 differentbreeds.

Interestingly, heterozygous dogs donot present symptoms that is similar tothe situation observed in Krt16-nullmice (Lessard and Coulombe, 2012).This feature is also observed in othermutated keratin recessive disorders,such as the epidermolytic palmop-lantar keratoderma Krt9� /� mousemodel (Fu et al., 2014), KRT10 muta-tions in humans and dog epidermolytichyperkeratosis (Credille et al., 2005;Muller et al., 2006; Gutierrez et al.,2013), or KRT14 mutations in epider-molysis bullosa simplex patients (Titeuxet al., 2011). These examples of rarerecessive forms show that, in the carrierpopulation, the healthy allele couldcompensate for the loss of expressionof the mutated allele, suggesting amechanism different from haploinsuffi-ciency usually reported in dominantinheritance.

qRT-PCR analysis of KRT16 mRNAexpression demonstrated a strongreduction (80%) in affected footpads(Supplementary Figure S1b online). Thisobservation is concordant with theabsence of the K16 protein as observedby immunostaining (Figure 2). As sug-gested by other studies, loss of onekeratin alters the expression patterns ofother plantar keratins, notably in res-ponse to skin injury (DePianto and

Coulombe, 2003; Lessard et al., 2013).These keratins could form an atypicalheterodimer between the partner of themutated keratin (here, K6a), and othertype I keratins, leading to the hyperpro-liferative phenotype (Muller et al., 2006).In the Dogue de Bordeaux keratoderma,KRT14 mRNA levels and K6 distribution,as observed by immunostaining, werenot significantly altered and otherkeratins are under investigation.

Spontaneous models such as dogbreeds that fully reproduce human dis-eases prove useful in understanding thegenetics and the physiopathology ofthese diseases and can lead to thedevelopment of efficient new therapies.

ETHICS STATEMENT

The work with dog samples wasapproved by the CNRS ethical board,France (35-238-13), for UMR 6290.Blood and tissue biopsy samples andpedigrees from dogs were collected by anetwork of veterinarians through theCaniDNA biobank developed by CNRSin 2000 (http://dog-genetics.genoues-t.org). The dog owners consented tothe use of data for research purposesanonymously.

CONFLICT OF INTERESTThe authors CK, CDdC, AT, and GQ are employ-ees of Antagene, a private company selling diag-nostic tests in dogs. The remaining authors state noconflict of interest.

ACKNOWLEDGMENTSWe are grateful to the Brittany Region (France; PhDfunding for JP), the European FP7 LUPA project(GA-201370), the Centre National de la RechercheScientifique (CNRS), and the French Society of theDogue de Bordeaux breed (SADB) with the FrenchKennel Club SCC (Societe Centrale Canine) forfunding and to the veterinarians, breeders, and todog owners who provided samples, pedigree, andclinical data; we especially thank B. Hedan, C. DeBrito, M. Heuze, R. Lavoue, X. Langon, T. Bord, P.Autret, and E. Voisin for clinical data. Finally, wewarmly thank S. Cure for English corrections andher kind availability.

Jocelyn Q1Plassais1,2, Eric Guaguere3,Laetitia Lagoutte1,2,Anne-Sophie Guillory1,2,Caroline Dufaure de Citres4,Frederique Degorce-Rubiales5,Maxence Delverdier6,Amaury Vaysse1,2,7,8, PascaleQuignon1,2, Celine Bleuart6,Christophe Hitte1,2, Alain Fautrel9,Cecile Kaerle4, Pascale Bellaud10,

Emmanuel Bensignor11,Guillaume Queney4,Emmanuelle Bourrat12,Anne Thomas4,13 andCatherine Andre1,2,13

1CNRS, UMR 6290, Institut de Genetique etDeveloppement de Rennes, Rennes, France;2Universite Rennes 1, UEB, Biosit, Faculte deMedecine, Rennes, France; 3CliniqueVeterinaire Saint Bernard, Lomme, France;4Antagene, Animal Genetics Laboratory, LaTour de Salvagny, France; 5Laboratoired’Anatomie Pathologique Veterinaire du Sud-Ouest LAPVSO, Toulouse, France; 6Serviced’Anatomie P Q2athologique, Ecole Veterinaire deToulouse, Toulouse, France; 7INSERM, UMR946, Genetic Variation and Human DiseasesUnit, Paris, France; 8Universite Paris Diderot,Sorbonne Paris Cite, Institut Universitaired’Hematologie, Paris, France; 9INSERM, UMR991, Universite de Rennes 1, BiositBiogenouest, Rennes, France; 10Universite deRennes1, Plateforme H2P2, Biosit Biogenouest,service d’anatomie pathologiques, Rennes,France; 11Clinique Veterinaire de la Boulais,Cesson-Sevigne, France and 12Departement deDermatologie, Hopital Saint-Louis, Paris, FranceE-mail: catherine.andre@univ-rennes1.fr13These authors contributed equally to this work.

SUPPLEMENTARY MATERIAL

Supplementary material is linked to the onlineversion of the paper at http://www.nature.com/jid

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