Association of non-synonymous variants with DWLM.

<p>ᵃ Parents of affected dogs were classified as obligate carriers.</p><p>^b These dogs consist of 47 control dogs with whole genome sequences (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108917#pone.0108917.s001" target="_blank">S1 Table</a>) and 499 control dogs that were specifically genotyped for the <i>VLDLR</i>:<i>c</i>.<i>1713delC</i> variant (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108917#pone.0108917.s002" target="_blank">S2 Table</a>). One of the whole genome sequences (sample BC273) did not have any coverage at the <i>VLDLR</i>:<i>c</i>.<i>1713delC</i> and the <i>PPAPDC2</i>:<i>c</i>.<i>202G>A</i> variant.</p><p>^c The 9 cases carried identical homozygous marker haplotypes in the critical interval. Thus the heterozygous genotype at this position in one of the cases was quite unexpected. We speculate that it may be due to an ancestral gene conversion event or, alternatively, a <i>de novo</i> revertation mutation.</p><p>Association of non-synonymous variants with DWLM.</p

Association of non-synonymous variants with DWLM.

<p>ᵃ Parents of affected dogs were classified as obligate carriers.</p><p>^b These dogs consist of 47 control dogs with whole genome sequences (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108917#pone.0108917.s001" target="_blank">S1 Table</a>) and 499 control dogs that were specifically genotyped for the <i>VLDLR</i>:<i>c</i>.<i>1713delC</i> variant (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108917#pone.0108917.s002" target="_blank">S2 Table</a>). One of the whole genome sequences (sample BC273) did not have any coverage at the <i>VLDLR</i>:<i>c</i>.<i>1713delC</i> and the <i>PPAPDC2</i>:<i>c</i>.<i>202G>A</i> variant.</p><p>^c The 9 cases carried identical homozygous marker haplotypes in the critical interval. Thus the heterozygous genotype at this position in one of the cases was quite unexpected. We speculate that it may be due to an ancestral gene conversion event or, alternatively, a <i>de novo</i> revertation mutation.</p><p>Association of non-synonymous variants with DWLM.</p

Variants detected by whole genome re-sequencing of an affected Eurasier.

<p>^a The sequences were compared to the reference genome (CanFam 3) from a Boxer. Only homozygous variants are reported.</p><p>Variants detected by whole genome re-sequencing of an affected Eurasier.</p

Four non-synonymous variants in the critical interval of an affected Eurasier that were absent from 47 other dog genomes.

<p>Four non-synonymous variants in the critical interval of an affected Eurasier that were absent from 47 other dog genomes.</p

Mapping of DWLM in Eurasier dogs.

<p>(A) A genome-wide association study using 9 cases and 11 controls indicates a signal with multiple associated SNPs on chromosome 1. The p-values are inflated due to the use of closely related animals. The inserted quantile-quantile (qq) plots show the observed versus expected log p-values. The straight black line in the qq-plots indicates the distribution of SNP markers under the null hypothesis. The straight red line visualizes the inflation of p-values caused by the close relatedness. This inflation is randomly distributed across the entire genome. The skew at the right edge of the genome-wide qq-plot indicates that several markers on chromosome 1 are stronger associated with DWLM than it would be expected by chance. This skew is absent when chromosome 1 markers are omitted from the plot. (B) The detailed view of chromosome 1 suggests an associated interval of approximately 10 Mb at ~91–101 Mb. (C) Homozygosity mapping. Each horizontal bar corresponds to one of the 9 analyzed cases. Homozygous regions with shared alleles are shown in blue. A shared homozygous interval of ~3.4 Mb delineates the exact boundaries of the critical interval from 90,860,923 bp to 94,212,001 bp (CanFam 3 assembly).</p

Experimental confirmation of the DWLM associated cytosine deletion by Sanger sequencing.

<p>(A) Electropherograms of the <i>VLDLR</i>:<i>c</i>.<i>1713delC</i> variant. A fragment harboring exon 12 and flanking sequences of the <i>VLDLR</i> gene was PCR-amplified and sequenced with the Sanger method. The figure shows representative traces from a normal and a DWLM affected Eurasier dog. The position of the deleted cytosine is indicated by an arrow. (B) Perfect cosegregation of the <i>VLDLR</i>:<i>c</i>.<i>1713delC</i> variant with the DWLM phenotype in four litters of Eurasier dogs. Filled symbols represent DWLM affected dogs. Obligate carriers are indicated by half-filled symbols. The transmission of the two different alleles is within the expected equal ratio and the offspring’s genotype distribution corresponds to Mendelian rules.</p

Biological processes enriched by RAO-regulated genes.

<p>The bars represent the number of RAO-regulated genes by the overall RAO-effect, regardless stimulation and/or cohort.</p

Variants detected by whole genome re-sequencing of an affected Kromfohrländer.

a<p>The sequences were compared to the reference genome (CanFam 3.1) from a Boxer. Only variants that were homozygous in the affected Kromfohrländer are reported.</p

Workflow of the analysis.

<p>The principal steps of the analysis and the format of the output files are given.</p

Clinical phenotype of digital hyperkeratosis.

<p>(A) Paw of a 1 year old affected Kromfohrländer. Note the cracked surface and deep fissures of the foot pads. (B) Paw of a control Kromfohrländer. (C) Hair coat of the dogs shown in panels A and B. The affected dog (left) has a more irregular coat appearance in comparison to the unaffected control dog (right). Both dogs are representatives of the wire-haired (“rough-coated”) Kromfohrländer variety.</p