A de novo variant in the keratin 1 gene (KRT1) in a Chinese shar-pei dog with severe congenital cornification disorder and non-epidermolytic ichthyosis.

A 3-months old Chinese shar-pei puppy with ichthyosis was investigated. The dog showed generalized scaling, alopecia and footpad lesions. Histopathological examinations demonstrated a non-epidermolytic hyperkeratosis. The parents of the affected puppy did not show any skin lesions. A trio whole genome sequencing analysis identified a heterozygous de novo 3 bp deletion in the KRT1 gene in the affected dog. This variant, NM_001003392.1:c.567_569del, is predicted to delete a single asparagine from the conserved coil 1A motif within the rod domain of KRT1, NP_001003392.1:p.(Asn190del). Immunohistochemistry demonstrated normal levels of KRT1 expression in the epidermis and follicular epithelia. This might indicate that the variant possibly interferes with keratin dimerization or another function of KRT1. Missense variants affecting the homologous asparagine residue of the human KRT1 cause epidermolytic hyperkeratosis. Histologically, the investigated Chinese shar-pei showed a non-epidermolytic ichthyosis. The finding of a de novo variant in an excellent functional candidate gene strongly suggests that KRT1:p.Asn190del caused the ichthyosis phenotype in the affected Chinese shar-pei. To the best of our knowledge, this is the first description of a KRT1-related non-epidermolytic ichthyosis in domestic animals.

Introduction

Ichthyoses are a heterogeneous group of hereditary cornification disorders. They are characterized by generalized dry skin, scaling and/or hyperkeratosis. Several genetically distinct forms have been identified in a variety of dog breeds [1]. An epidermolytic form with autosomal recessive inheritance due to a variant in epidermal keratin 10 (KRT10) has been documented in the Norfolk terrier [2]. Six other canine ichthyosis forms that are characterized at the molecular level represent non-epidermolytic ichthyoses. A PNPLA1-associated autosomal recessive form that involves altered glycerophospholipid metabolism has been reported in golden retrievers [3, 4]. A loss of function variant in the TGM1 gene encoding transglutaminase 1 leads to autosomal recessive ichthyosis in Jack Russell terriers due to calcium dependent cross-linking of peptides (e.g. involucrin, loricrin) involved in forming the cornified envelope [5]. NIPAL4 (ichtyn) deficiency was reported in ichthyotic American bulldogs [6, 7]. ABHD5-related autosomal recessive ichthyosis represents another defect in lipid metabolism that has been reported in golden retrievers [8]. An autosomal dominant form of ichthyosis in a German shepherd dog was caused by a missense variant in ASPRV1 encoding a protease required for the posttranslational processing of profilaggrin [9]. Finally, an autosomal recessive SLC27A4-related severe syndromic form of ichthyosis has been reported in Great Danes [10, 11]. Moreover, cornification disorders suggestive of ichthyosis have been described based on clinical examination and histopathologic changes in soft-coated wheaten terriers, West Highland white terriers, English springer spaniels, Labrador retrievers [1].

While cases of KRT1-related ichthyosis have been documented in humans, they have not been reported in dogs to date [12-14]. This investigation documents a congenital cornification disorder in a Chinese shar-pei puppy due to a 3 base pair deletion in the KRT1 gene.

Materials and methods

Clinical examinations

Clinical evaluation of the patient was performed by a board certified veterinary dermatologist (TN). Skin scrapings and skin cytology were performed, and punch biopsies from the right antebrachium, the neck and right shoulder were collected for histopathologic and immunohistochemical examination. Blood samples from patient and his parents were collected for genetic testing.

Histopathological and immunohistochemical examinations

Submitted formalin fixed punch biopsies were bisected. Four-micron, hematoxylin and eosin-stained paraffin-sections and immunohistochemical stains were evaluated by a board certified veterinary pathologist (VKA).

Immunohistochemistry for KRT1 expression was performed on all three biopsy samples from the patient as well as on sections healthy skin from two Chinese shar-pei dogs (and other breeds (standard poodle, terrier-mix, boxer)). Four micron paraffin sections were collected on “plus” coated slides and air dried at 37°C overnight and subsequently deparaffinized (xylene: 10 min 2x, followed by 100% ethanol: 1 min 3x, 95% ethanol: 1 min and 70% ethanol: 1min). After quenching of endogenous peroxidase (500 ul 10% sodium azide; 500 ul 30% hydrogen peroxide in 50 ml PBS; 25 min at room temperature), slides were rinsed in PBS 3x and immersed in preheated antigen retrieval solution (1x Dako Target Retrieval Solution; stock solution S1699, pH6); retrieval was performed a pressure cooker for 5 minutes. Slides were cooled down to room temperature, washed in PBS 3x. After exposing slides to 10% horse serum in PBS (15 min) the anti-CK-1 antibody (Clone 4D12B3: sc-65999; Santa Cruz Biotechnology, Inc. Dallas, Texas USA; 1:500 dilution in 10% horse serum in PBS) was applied for 60 min. After three rinses in PBS the following steps were performed: application of ImmPRESS HRP Horse Anti-Mouse IgG Polymer Reagent (Vector Cat.# MP-7402; 30 min), thorough PBS rinses and addition of substrate (Vector, SK-4800). Development was monitored microscopically and reaction was stopped by immersing the slides in Milli-Q/distilled water. Counterstain (Gill’s Hematoxylin #2 RICCA, 3536–16; 15–30 s) was stopped by washing slides in running tap water. Slides were then cover-slipped using Shandon-Mount media (Thermo Scientific, 1900331).

Genetic examinations

Animal selection

This study included a total of 22 Chinese shar-peis. They comprised one ichthyosis affected Chinese shar-pei and its unaffected parents. Additional samples from 19 unrelated Chinese shar-peis without clinical signs of ichthyosis from the Vetsuisse Biobank were used as controls.

Whole-genome sequencing

Illumina TruSeq PCR-free libraries with insert sizes of ~330 bp were prepared from the affected dog and both parents. The libraries were sequenced with 2 x 150 bp chemistry on a NovaSeq 6000 instrument. The reads were mapped to the CamFam3.1 reference genome assembly as described [15]. The sequence data were submitted to the European Nucleotide Archive with the study accession PRJEB16012 and sample accessions SAMEA7198604 (affected puppy), SAMEA7198605 (unaffected dam) and SAMEA7198612 (unaffected sire). Variant calling was performed as described [15]. To predict the functional effects of the called variants, the SnpEff software [16] together with NCBI annotation release 105 for the CanFam 3.1 genome reference assembly was used. For variant filtering, we used 793 control genomes derived from 784 dogs and 9 wolves (S1 Table). We applied two different hard filtering approaches for homozygous and heterozygous private variants in the affected dog: In the search for private homozygous variants, we retained only variants with genotype 1/1 in the affected puppy and genotypes 0/0 or ./. in the 793 control genomes. In the search for private heterozygous variants, we retained only variants with genotype 0/1 in the affected puppy and genotypes 0/0 or ./. in the 793 control genomes. Subsequently, the private variants were combined in an Excel-file for further inspection (S2 Table). For functional prioritization, variants with SnpEff impact predictions high or moderate were combined and termed "protein-changing variants".

Confirmation of parentage

To confirm the parentage of the presumed parents and the affected dog, we used the genome sequence data (vcf-file) of the affected dog and its parents. Using PLINK v1.9 we extracted 6,269,532 informative markers distributed over all autosomes and performed a pairwise IBD estimation with the—genome command [17]. The sire-offspring and dam-offspring pairs both had an estimated overall IBD proportion (PI_HAT) of 50% with 0% P(IBD = 0), 100% p(IBD = 1) and 0% p(IBD = 2) as expected for parent-offspring duos.

Gene analysis

We used the dog reference genome assembly CanFam3.1 and NCBI annotation release 105. Numbering within the canine KRT1 gene corresponds to the NCBI RefSeq accession numbers NM_001003392.1 (mRNA) and NP_001003392.1 (protein). For a multiple species comparison of KRT1 amino acid sequences, we used these accessions: NP_006112.3 (Homo sapiens), NP_001104288.1 (Pan troglodytes), XP_002687292.1 (Bos taurus), NP_032499.2 (Mus musculus), NP_001008802.2 (Rattus norvegicus). A precomputed multiple species sequence alignment was obtained from the NCBI HomoloGene website (https://www.ncbi.nlm.nih.gov/homologene).

Sanger sequencing

We used Sanger sequencing to confirm the KRT1:c.567_569del variant and to perform targeted genotyping of all samples. AmpliTaqGold360Mastermix (Thermo Fisher Scientific, Waltham, MA, USA) and the primers 5‘-CCT GGT GGC ATA CAG GAA GT-3‘ (forward primer) and 5‘-CTC GTT CGC ACC CTA GAA AG-3‘ (reverse primer) were used to amplify a 454 bp product. After treatment with shrimp alkaline phosphatase and exonuclease I, PCR amplicons were sequenced on an ABI 3730 DNA Analyzer (Thermo Fisher Scientific). Sanger sequences were analyzed using the Sequencher 5.1 software (GeneCodes, Ann Arbor, MI, USA).

Ethics statement

All animal experiments were performed according to the local regulations. The dogs in this study were privately owned and skin biopsies and blood samples for diagnostic purposes were collected with the consent of the owner. The collection of blood samples from healthy dogs was approved by the “Cantonal Committee for Animal Experiments” (Canton of Bern, Switzerland; permit 71/19).

Results

Family anamnesis, clinical examinations, histopathology

A 3-months old male Chinese shar-pei was presented for scaly skin and reduced overall body growth when compared with his 3 littermates, a female and 2 male puppies with clinically normal skin. Dam and sire were in the same household and clinically normal. Administration of Clavamox (ZoetisUS; 62.5 mg twice daily for 10 days) followed by Convenia (ZoetisUS; 80 mg/ml 0.54 ml), prednisone (5 mg twice daily, then once daily) and frequent bathing with HexaChlor-K shampoo (GelnHaven Therapeutics, Schuyler, Oregon) revealed minimal improvement. Terramycin eye ointment (ZoetisUS) had been applied for entropium of the left eye. At time of presentation, the dog appeared bloated and uncomfortable despite eating and consuming normal amounts of water.

At the time of presentation severe generalized scaling and alopecia was noted, with scaling most prominent on the head (Fig 1A), neck (Fig 1B), abdomen (Fig 1C), legs, axillary folds (Fig 1D) and paws. Prominent follicular fronds accompanied surface scaling. The paw pads appeared deformed and hyperkeratotic. Pruritus was not observed. The left eye had an entropium. Skin scrapings for Demodex mites were negative. Skin cytology revealed numerous yeast organisms.

Fig 1

Clinical presentation of affected Chinese shar-pei.

Severe generalized alopecia and scaling with marked follicular fronds on (A) head, (B) neck, (C) abdomen and (D) axillary folds.

Biopsies from all three locations revealed severe hyperkeratosis, characterized by prominent keratin lamellae overlaying a marked compact layer of keratin (Fig 2A and 2B). The epidermis was markedly acanthotic and most infundibular regions were markedly dilated resulting in narrowing of the interfollicular epidermis (Fig 2A). The follicular lumina were filled with keratin and the infundibular epithelium was hyperplastic. Some perinuclear clearing was most evident in the prominent granular layer with irregularly sized keratohyalin (Fig 2C). Dispersed mast cells and some plasma cells and neutrophils were present in the superficial dermis and the sebaceous glands were prominent. Several small neutrophilic crusts with some cocci were noted entrapped within the thick keratin layer (Fig 2D). In the sample from the shoulder some follicles contained neutrophils in their lumina and the epidermis was covered by parakeratosis. Superficial yeast organisms were not observed in sections stained with periodic acid-Schiff stain. Many hair follicles and remaining hair shafts contained clumped melanin. The following morphologic diagnoses were made: 1) severe acanthosis and superficial and follicular hyperkeratosis suggestive of a cornification disturbance and 2) multifocal neutrophilic pustular dermatitis and neutrophilic luminal folliculitis and 3) melanin pigment clumping indicating dilute hair coat color. The latter was considered an expected incidental finding as the dog had a d/d genotype at the MLPH gene [18, 19] and was born out of two clinically inconspicuous dilute-colored parents.

Fig 2

Histopathologic changes in the affected Chinese shar-pei.

(A and B) The epidermis is severely hyperplastic with extensive compact and lamellar hyperkeratosis of the surface. The hyperkeratosis extends into the follicular lumina dilating the follicular openings (H&E; 40x). (C) There is a prominent granular layer with irregularly sized keratohyalin granules and common perinuclear vacuoles (H&E; 400x). (D) Multifocally, there were neutrophilic crusts indicating secondary pyoderma.

Given the overwhelming features of follicular and superficial hyperkeratosis, a hereditary cornification disorder consistent with ichthyosis was considered. Pustules and superficial folliculitis indicated a secondary pyoderma, which, based on skin cytology, was accompanied by a superficial yeast infection.

Genetic analysis

In order to characterize the underlying causative genetic variant we sequenced the genome of the affected puppy at 18.9x coverage and searched for variants in 36 candidate genes for ichthyosis (S2 Table), which were exclusively present in the affected dog and absent from 793 control genomes (Tables 1 and S1 and S3).

Table 1

Variants detected by whole genome sequencing of the affected Chinese shar-pei.

Filtering step	heterozygous variants	homozygous variants
Variants in the whole genome	4,261,447	2,920,513
Private variantsa	82,046	9,522
Private protein-changing variantsa	503	39
Private protein-changing variants in 36 candidate genesa	2	0

aThe parents of the affected dog were excluded for these filtering steps.

Subsequently, we performed a trio analysis and compared the genotypes in the affected dog with the genotypes of both parents (S3 Table). We considered two alternative scenarios for the putative causal variant: For an autosomal recessive trait, we expected the affected dog to be homozygous for the alternate allele and both parents heterozygous. Alternatively, for a dominant trait that could only have been caused by a de novo mutation event, the affected dog should be heterozygous and both parents should be homozygous for the reference allele. The results of the trio analysis are summarized in Table 2.

Table 2

Trio analysis of the affected Chinese shar-pei and its parents.

Filtering step	heterozygous variants	homozygous variants
Private variants that were absent from 793 control genomes	82,046	9,522
Protein-changing & genotypes of parents compatible with a pathogenic effect	27	19
Protein-changing & genotypes of parents compatible & in 36 candidate genes	1	0

Taken together these analyses identified a single protein-changing variant in a known ichthyosis candidate gene, for which the genotypes of the parents were compatible with a pathogenic effect. The variant was a heterozygous in frame deletion in the first exon of KRT1 (NM_001003392.1:c.567_569del) (Fig 3A), removing three nucleotides coding for an asparagine of the 1A coil domain (NP_001003392.1:p.(Asn190del), Fig 3B). The formal genomic designation of the variant is Chr27:2,422,716_2,422,718del (CanFam3.1).

Fig 3

Details of the KRT1:c.567_569del, p.(Asn190del) variant.

(A) Integrative Genomics Viewer (IGV) screenshot showing the short-read alignments of the ichthyosis affected puppy and its non-affected parents at the position of the deletion. A deletion of one copy of the allele is visible in the case but not in the parents. Note that in the IGV screenshot bases 2,422,713–2,422,715 are deleted, whereas the 3’-rule of HGVS nomenclature requires to designate this variant as Chr27:2,422,716_2,422,718del (CanFam3.1). (B) Schematic representation of the protein domain structure of a keratin dimer [20] with the highly conserved amino acid sequence of the coil 1A subdomain shown below. The variant is predicted to delete an asparagine residue from coil 1A, which is located within the rod domain of KRT1.

The trio analysis comparing the variants in the affected dog with the genomes of both parents revealed that KRT1:c.567_569del represented a de novo variant as the mutant allele was absent from leukocyte DNA of both parents. The correct parentage of sire and dam in this family was confirmed based on the genome sequence data.

We used Sanger sequencing to confirm the identified candidate KRT1: c.567_569del variant and to genotype the rest of the Chinese shar-peis from our study. The deletion was only present in heterozygous state in the ichthyosis affected puppy whereas both parents and all remaining Chinese shar-peis were homozygous for the wild type allele.

Expression of KRT1

Given the de novo variant in the KRT1 gene in this Chinese shar-pei, KRT1 expression of the tissue was evaluated by light microscopy using immunohistochemistry. The intensity of KRT1 expression in the epidermis and the follicular epithelia of the affected dog was visually comparable to normal skin samples from Chinese shar-pei dogs (Fig 4) and other breeds. Keratinocytes revealed strong membranous and cytoplasmic KRT1 expression.

Fig 4

KRT1 expression in Chinese shar-pei skin (immunohistochemistry with anti-KRT1 antibody).

(A) Intensity of KRT1 expression in the epidermis and follicular epithelia of the affected Chinese shar-pei is comparable to (B) normal Chinese shar-pei skin (40x). (C) Strong KRT1 expression in the epidermis of the affected Chinese shar-pei (400x).

Discussion

Reported canine hereditary cornification disorders are due to genetic variants affecting either keratins or components involved in crosslinking of peptides or disruption of lipids within the cornified envelope [1]. All of them lead to interruption of successful cornification or desquamation. Most well documented forms of canine ichthyosis are non-epidermolytic, involving genetic variants in ABHD5 or PNPLA1 in golden retrievers, TGM1 in Jack Russell terriers, NIPAL4 in American bulldogs, and ASPRV1 in a German shepherd dog [3–11, 21].

Marked follicular fronds in addition to prominent surface scales observed in this Chinese shar-pei indicated a cornification disturbance affecting both epidermis as well as follicles. Follicular fronds appear to be less prominent in other breeds with non-epidermolytic ichthyosis [3, 5, 6, 21].

Associated with the marked epidermal and follicular hyperkeratosis was a rather prominent acanthosis. The latter was also present in areas with no evidence of secondary pyoderma. This is somewhat in contrast with non-epidermolytic ichthyosis in other dog breeds, where marked hyperkeratosis is often disproportionate to the degree of epidermal acanthosis [5, 6, 21]. However, the marked hypergranulosis with irregularly sized keratohyalin granules as well as the presence of mild perinuclear vacuolization of keratinocytes was a finding consistent with features seen in other breeds. Electron microscopy revealed curvilinear membranous material within the granular layer cytosol, also in particular in the perinuclear swellings seen on light microscopy [6]. Electron microscopic evaluation was not performed in the Chinese shar-pei investigated by us.

Genetic variants in KRT1 or KRT10 in humans typically present with an epidermolytic ichthyosis [12, 22–24]. The KRT1-related epidermolytic hyperkeratosis presents with or without palmo-plantar keratoderma, while KRT10-related epidermolytic ichthyosis typically does not involve palmo-plantar keratoderma. A KRT10 variant in Norfolk terriers results in epidermolytic ichthyosis [2], but the KRT1 variant in the Chinese shar-pei of this investigation was not associated with epidermolytic changes. Altered KRT1/KRT10 dimer formation due to variants affecting the structure of the paired 2B and V2 domains leads to severe acanthosis and hyperkeratosis in humans [25], reflecting the histologic features observed in the affected Chinese shar-pei. In human literature this is also referred to as “epidermolytic ichthyosis sine epidermolysis” [26].

The identified variant in the affected Chinese shar-pei leads to a deletion of 3 bases coding for an asparagine in the coil 1A motif of KRT1, which is part of the central rod domain of the KRT1/KRT10 heterodimer. The sequence of the central rod domain is highly conserved amongst epidermal keratins [20]. Three different missense variants affecting the homologous asparagine-188 of KRT1 in human patients have previously been described to cause epidermolytic hyperkeratosis [22, 27, 28]. This strongly suggests that changes in this region are intolerable for functional keratin filament formation.

In the affected Chinese shar-pei, the mutant KRT1 protein lacking asparagine-190 is apparently expressed within the acantholytic epidermis and the follicular epithelia as demonstrated by the immunohistochemistry experiment. The expression level was comparable to control skin tissue from an unaffected Chinese shar-pei and dogs from other breeds. This might indicate that the single amino acid deletion possibly interferes with dimerization or another function of the KRT1 molecule.

Our genetic analysis revealed KRT:p.(Asn190del) as a highly plausible candidate causal variant for the observed phenotype. According to the ACMG/AMP consensus standards for the interpretation of sequence variants in human patients [29], our data provide one strong, three moderate and one supporting criteria for pathogenicity, which is sufficient to classify the variant as pathogenic. The strong criterion is the demonstration of a de novo variant in an affected dog born out of two healthy parents. The three moderate criteria are the absence of the mutant allele from a relatively large control cohort, the protein length change due to an in-frame deletion and the fact that missense variants affecting the same amino acid have been established as pathogenic in humans. We consider the highly specific disease phenotype with known monogenetic etiology as supporting criterion for pathogenicity. Although our analysis yielded a variant fulfilling diagnostic criteria for pathogenicity, we have to caution that we did not investigate structural variants. Furthermore, the analysis relied on the accuracy and completeness of the CanFam3.1 genome assembly and NCBI annotation release 105. Therefore, we cannot formally exclude the possibility that other potentially plausible variants were missed. NCBI annotation release 105 agrees with a manually curated annotation of the canine KRT1 gene [30].

In summary, to the best of our knowledge, this is the first case of a KRT1-related ichthyosis reported in domestic animals and the first case of ichthyosis in a Chinese shar-pei dog.

Accession numbers of 796 dog/wolf genome sequences.

The affected dog is highlighted in red and the non-affected parents are highlighted in blue. Both parents and the affected dog were used for a trio analysis. The other 793 genome sequences were used as controls in filtering for private variants.

(XLSX)

Click here for additional data file.

36 candidate genes for ichthyosis.

List of non-syndromic and syndromic forms of ichthyosis.

(XLSX)

Click here for additional data file.

Private variants in the affected Chinese shar-pei.

Variants are listed multiple times, if they have predicted effects on more than one transcript. Variants with a SnpEff predicted impact of "high" or "moderate" were considered protein-changing variants. For private variant filtering, the genomes of the case and 793 controls (excluding the parents) were considered. The KRT1:c.567_569del variant is highlighted in yellow (line 176,297).

(XLSX)

Click here for additional data file.

12 Apr 2022

A de novo variant in the keratin 1 gene (KRT1) in a Shar Pei dog with severe congenital cornification disorder and non-epidermolytic ichthyosis

PLOS ONE

Dear Dr. Affolter,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Editor and reviewers find the manuscript to be largely sound and worthy of publication, however numerous inconsistencies in language and clarity need to be addressed as detailed in the reviewer comments.

Please submit your revised manuscript by May 27 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

4 Jun 2022

Responses to Reviewers:

„A de novo variant in the keratin 1 gene (KRT1) in a Chinese shar pei dog with severe congenital cornification disorder and non-epidermolytic ichthyosis”.

PONE-D-21-30440

We would like to thank the reviewers for the insightful suggestions. Please find our responses and comments listed below.

Sincerely,

Verena K. Affolter

Dr.med.vet., Dipl. ECVP, PhD

Professor of Clinical Dermatopathology

Chief of Service Anatomic Pathology

phone: (530) 754 0104; fax: (530) 752 3349

vkaffolter@ucdavis.edu

Reviewer 1:

(1)

Minor corrections

- Throughout, a consistent scheme for capitalization of dog breed names needs to be followed. The generally accepted convention is to capitalize only the proper nouns in the name. Likewise, the official breed names should be used when relevant.

o Throughout: Shar Pei = Chinese shar pei

Response: this has been corrected throughout the document

o Pg 3, line 49: Golden retriever = golden retriever

o Pg 3, line 54-55: German shepherd = German shepherd dog

o Pg 3, line 58: soft-coated Wheaten terriers = soft-coated wheaten terriers

o Pg 3, line 59: West Highland White terriers = West Highland white terriers

o Pg 3, line 59: English Springer spaniels = English springer spaniels

o Pg 4, line 78: Standard Poodle = standard poodle

o Pg 4, line 78: Terrier-mix = terrier mix

o Pg 4, line 78: Boxer = boxer

o Pg 10, line 244: German shepherd = German shepherd dog

o Pg 11, line 264: Norfolk Terriers = Norfolk terriers

Response: these have been corrected throughout the document

- Pg 3, line 61: people = humans Response: corrected; line 76

- Pg 3, line 67: board-certified dermatologist = board certified veterinary dermatologist Response: corrected; line 107

- Pg 4, line 74: board-certified pathologist = board-certified veterinary pathologist

Response: corrected; line 116

- Pg 4, line 77: “… three biopsy samples of the patient …” = “… three biopsy samples from the patient …” Response: corrected

- Pg 5, line 112: “… 793 control genomes (S1 Table).” Tell us roughly what the control genomes are. “… consisting of ### domestic dogs and ### wild canids.”

Response: text gas been adjusted; lines 162-170

- Pg 6, line 145: “… presented for scaly skin and reduced growth when …” Growth of what? Hair, skin, body size? Response: adjusted

- Pg 7, lines 151 – 152: should read “At the time of presentation, the dog appeared …” Response�  corrected

- Pg 7, line 159: “… for Demodex mites was negative.” Should be “… for Demodex mites were negative.” Response: corrected

- Pg 10, line 241: What is the envelope? Response: corrected to “cornified envelope”

- Pg 10, line 241: Add a period after “desquamation”. Response: corrected

- Pg 10, line 257: “Electron microscopic revealed …”. Do you mean “electron microscopy” or is there a missing word after “microscopic”? Response: corrected

- Pg 11, line 259: Remove the “; it” at the end of the line. Response�  corrected

- Pg 11, line 261: people = humans. Response: corrected

- Pg 11, line 268: Should read “… reflecting the histologic features observed in the affected Chinese shar pei.” Response: corrected

- Pg 12, lines 286 – 288: The last sentence does not belong here. Move it up somewhere into the body of the Discussion. Response: changes has been made

(2)

Moderate to substantial corrections

In the introduction, add 1 – 2 sentences describing “hereditary cornification disorders” and “ichthyosis”.

Response: Revised accordingly

(3)

Pg 8, line 180 mentions that dilute hair coat color was considered incidental to the skin findings. None of the candidate genes investigated later were related to coat color or associated hair/skin conditions. Should any consideration be given to the possible overlap with color dilution alopecia, which is commonly seen with the “D locus” dilute coloration?

Response: We thank the reviewer for this important comment. Both parents of the affected puppy were also dilute-colored and did not show clinically manifest color dilution alopecia. Color dilution in various dog breeds presents with very mild follicular hyperkeratosis and may or may not be associated with alopecia. Moreover, alopecia due to color dilution is a gradually developing process, typically presenting clinical signs over a period of years. We therefore think that any potential contribution of the coat color dilution to the phenotype in the ichthyotic puppy is minimal at most. We added a short statement about the parents to this section.

(4)

Why were only known ichthyosis candidate genes considered? In the trio analysis, you identified 542 protein-changing variants, which is a very reasonable number of genes to investigate for possible functional implications. You may wind up with the same answer, I think you have sufficient evidence to suspect that the KRT1 variant is correct, but by not limiting yourself to a list of candidate genes, you are better able to make your case. What other genes were affected by the 542 protein-changing variants, and would any of them be potential functional candidates?

Response: We revised the results section and give a more detailed description of the results that hopefully better illustrates why our analyses ended up at a single candidate variant. We also revised the S3 table and give now all 91,568 private variants to allow the interested reader to evaluate other candidate genes and/or variants. We are afraid that due to space considerations, we don't think that a verbose discussion of the other genes and variants would be helpful.

(5)

- Table 1: What does “Genotypes of parents compatible with a pathogenic effect” mean? It sounds like there is one variant found in the parents that may be pathogenic? What variant is that, what gene?

Response: We agree with the reviewer that our initial presentation was confusing. We think that we have addressed this with the changes outlined under the previous comment.

Reviewer 2:

(1)

While this does appear to be a good candidate variant, there are numerous instances where the language describing the potential role is not appropriate based on the analyses yet completed. For example lines 34-35 (“…strongly suggests…caused”), line 63 (“…due to a 3 base pair…”), and line 276 (“This strongly suggest…”). The authors should rephrase these (and any other) passages to reflect potential association as opposed to causation.

Response: We applied the ACMG/AMG consensus standards for the interpretation of sequence variants in human patients (Richards et al. 2015 Genet Med 17:405-424) and extrapolated the human guidelines to dogs. Using this approach we have the following support for the causality of the KRT:p.Asn190del variant:

• PS2, de novo in a patient with the disease and no family history

• PM2, mutant allele is absent from controls

• PM4, protein length change due to in-frame deletion in a non-repeat region

• PM5, novel missense change (in our case a single amino acid deletion) where a different missense change determined to be pathogenic has been seen before

• PP4, patient's phenotype is highly specific for a disease with a single genetic etiology

Thus, we have 1 strong, 3 moderate and 1 supporting criteria that support the causality. According to human diagnostic standards, this is sufficient to classify the variant as pathogenic. We therefore think that our evidence is sufficient to claim causation. We chose a strong ("strongly suggests to cause") rather than an absolute ("causes") wording to take into account the cross-species extrapolation. We added a paragraph at the end of the discussion outlining this argumentation.

(2)

Line 31 (and the discussion) speculate that the deletion “interferes with keratin dimerization”. While this certainly could be true based on the previous literature, this has not been thoroughly investigated here. The immunohistochemistry presented herein does not suggest this really. If the authors want to include a description of this potential mechanism the language should be toned down (as in the first comment) and likely removed from the abstract. Alternatively, and if possible, it would be really interesting to see the results from some type of in-vitro protein-protein interaction assay.

Response: We agree with the reviewer that our manuscript lacks experimental support for the proposed defect in keratin dimerization. We modified the statement in the abstract and the discussion, which now reads: "This might indicate that the variant possibly interferes with keratin dimerization or another function of KRT1." We hope that it is acceptable to keep the proposed hypothetical pathomechanism in the abstract.

(3)

How was the variant filtering accomplished with the 793 public control genomes? In addition, I noticed there were no Shar Pei’s in the control set. Were there none available or were they excluded? A reference (14) was used to cover how the mapping and variant calling were conducted but additional descriptions of how the variant filtering was conducted, why these 793 were selected, and why no other healthy Shar Pei’s were included is needed.

Response: We added a description of our variant filtering process to the methods. The 793 control genomes represented a convenience sample that we also used for other similar projects in the past. It is an expanded version of the DBVDC dataset described in the reference Jagannathan et al. 2019. Preparing and handling such massive datasets with hundreds of mammalian genomes (~50 Tb raw sequencing data) takes many weeks of computing time at the high performance computing cluster of the University of Bern. While the inclusion of non-affected unrelated Shar Pei controls would have been desirable from a scientific point of view, the costs for the generation and analysis of such data would have been prohibitive for our study.

(4)

Why was confirmation of parentage done with VCFtools as opposed to a more commonly used parentage/genetic marker test? In this section they are referred to as “presumed parents” but “unaffected parents” earlier suggesting not presumed. Similarly, lines 211-212 state parentage was “confirmed based on the genome sequence data.” Please include clarification as to the why and how here – this is important so that we have absolute confidence that the parents are in fact the parents.

Response: We repeated the parentage confirmation and revised the methods section accordingly. There was no need to perform additional laboratory experiments as we had the full genome sequences of the trio consisting of the offspring and both parents. We extracted the genotypes at more than 6 million informative variants to confirm the parentage, which is "even better" than a routine parentage test that would be based on either 10-20 microsatellite markers or less than 300 single nucleotide variants. We hope that the revised methods section is now sufficiently detailed to clear all possible doubts about the true parentage.

(5)

Line 122 the authors should include a description of the multiple sequence alignment method.

Response: We added the requested method (reference to NCBI HomoloGene website).

(6)

The candidate gene set appears to be from three previous papers, one of which is for a dog with ichthyosis. Were there attempts to expand the search for candidate genes via databases such as OMIM or PubMed? Additional descriptions on why only these sources were used should be included. Perhaps a structured systematic review is not needed but a more complete explanation on how the candidate gene set was developed would be helpful in understanding why this severe filtering (92k to 500 variants) was needed.

Response: We think that it is a standard approach in medical genetics (for humans and animals) to focus on known functional candidate genes when single cases with presumed inherited diseases with characteristic phenotypes such as an ichthyosis are investigated. For our list of candidate genes, we compiled the information from two human review articles listing a total of 35 functional candidate genes. These two reviews are based on systematic reviews of OMIM and the scientific literature. We added ASPRV1 as this is an ichthyosis gene that was only recently discovered in dogs and subsequently in human ichthyosis patients and is not yet contained in the older reviews.

As our initial hypothesis-based functional candidate gene approach apparently worked, we don't think it is appropriate to provide a lengthy verbose justification of the selection of candidate genes. Such lists are always debatable. If the initial approach had been unsuccessful, we would of course have performed additional and possibly hypothesis-free analyses.

We revised the S3 table and now provide a full list of all private variants in the affected dog. Thus, interested readers have the opportunity to reproduce our analyses and/or to perform the filtering and prioritization steps of the ~7 million starting variants in different orders. We are convinced that, independent on the specific order of filtering steps, the KRT1 variant will always be identified as the most likely cause of the observed phenotype.

(7)

Table 1 lists 92k private variants, were there any attempts to examine this set for other potential variants compatible with an autosomal recessive inheritance pattern that could explain the phenotype? The definition of “protein-changing” should be probably be included somewhere as well. I would suggest including some type of analyses on the 92k private variants. For example, theses could be categorized by location, type, and predicted impact (I believe SnpEff outputs summary files containing this type of data?). Similar to the candidate gene comment above, the authors should examine at least the predicted high-impact variants for other potential genes that may be associated with keratinization disorder.

Response: We revised and expanded the methods and results section. When the genotypes of the parents and a protein-changing effect (SnpEff impact high or moderate) are taken into account, of the starting 92 k private variants only 46 remain and only 1 of those is in a functional candidate gene.

(8)

Lines 231 and 235 state expression was “comparable” between the affected and normal dogs. It looks a bit a subjective and to the untrained eye, these appear rather different on paper. It would seem that in order to make statements about comparability, protein quantification should be conducted. Transcript quantification via RNA-seq or qPCR may be warranted as well to uncover any potential differences in expression. Baring any additional experiments, perhaps there is a pathologist associated with this study that could describe how these were identified as comparable?

Response: Histology and immunohistochemistry has been reviewed by a board-certified veterinary pathologist specialized in dermatopathology and experienced in evaluating immunohistochemical stains. We revised the text section describing the results of the immunohistochemistry experiment.

(9)

There should probably be some mention in the discussion of the uncertainty inherent in using the public annotation as there are known issues. For example, does this deletion appear in the same amino acid and domain using the Ensembl annotation which lists two isoforms?

Response: We added a verbose statement on the limitations of our approach to the discussion. We also added a comment and a reference that the NCBI annotation that we used for the study corresponds to a manually curated annotation for the canine KRT1 gene. This is another example, where the NCBI annotation seems to be superior to the ENSEMBL annotation for the dog. We consider the ENSEMBL annotation for the canine KRT1 gene incorrect.

(10)

There is a discrepancy in author order between the cover sheet and manuscript. Please correct.

Response: the order of listed Authors is the same on cover letter as well as manuscript

(11)

Lines 46-57 is a single sentence and as a result a little difficult to follow. I would suggest breaking this up or restructuring it.

Response: We revised this long enumeration and built smaller sentences.

(12)

Table 1 legend “genome resequencing”. Is this supposed to be “genome sequencing” or was this dog previously sequenced?

Response: We used the term "resequencing" to indicate that we did not perform a de novo genome assembly for this dog, but rather aligned the short-read sequence data to an existing reference genome. While this is the technically correct term, we realize that it may be confusing to the non-specialist reader. Therefore, we now replaced it with "whole genome sequencing".

(14)

Figure 3 legend has a reference error {Bray, 2015 #17}.

Response: Revised accordingly.

(15)

Minor inconsistency in naming the protein dimer (line - 266 KRT1/10; line 273 - KRT1/KRT10). I am not sure which is correct but should be consistent I would think.

Response: We consistently revised the entire manuscript and use now KRT1 instead of keratin 1 or K1.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

15 Sep 2022

A de novo variant in the keratin 1 gene (KRT1) in a Chinese shar pei dog with severe congenital cornification disorder and non-epidermolytic ichthyosis

PONE-D-21-30440R1

Dear Dr. Affolter,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Gerhard Wiche, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have adequately addressed all of the points that I raised in the initial review. I am comfortable now recommending this manuscript for publication.

Reviewer #2: All comments/questions were addressed thoughtfully by the authors. The manuscript is technically sound and the underlying appears to be publicly available.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

**********

27 Sep 2022

PONE-D-21-30440R1

A de novo variant in the keratin 1 gene (KRT1) in a Chinese shar-pei dog with severe congenital cornification disorder and non-epidermolytic ichthyosis

Dear Dr. Affolter:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Prof. Gerhard Wiche

Academic Editor

PLOS ONE