Schematic overview of candidate causative mutations upstream of the canine <i>MITF-M</i> promoter and luciferase reporter activity.

<p>(A) The SINEC-Cf, SNP#21 and Lp sequences included in constructs are indicated together with a comparative human-dog sequence alignment over the region. Tracks representing 7X regulatory potential and mammalian conservation for the corresponding region in humans are indicated. The broken vertical lines indicate the border between the promoter region and the upstream region combined in individual constructs. The region −1400 to −2800 bp upstream of the <i>MITF-M</i> promoter was not included in the construct. (B) Overview of the six luciferase reporter constructs used to assess the regulatory potential of different combinations of the SINE, SNP#21 and Lp variants and results of reporter assays. (C) Critical elements of the canine <i>MITF-M</i> promoter. Schematic overview of the canine <i>MITF-M</i> minimal promoter. Three different insert fragments (1, 2 and 3) and two variants of each fragment were designed corresponding to the <i>S</i> and <i>s^w</i> haplotypes. The insert borders were defined based on the predicted transcription factor binding sites as indicated. Firefly luciferase reporter levels in B and C are presented in relation to control <i>Renilla</i> luciferase levels, normalized against the empty control vector. Stars in the graph indicate reporter activity significance levels in pair-wise comparisons; N.S. = Non Significant, * P<0.05, ** P<0.01, *** P<0.001. Error bars represent standard error of the mean. RLU = Relative Luciferase Units.</p

Polymorphisms included in the Luciferase reporter design defined in Figure 3.

<p>The positions of the SINE and length polymorphisms are indicated, as are positions of additional polymorphisms previously considered unlikely to be functionally important <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104363#pone.0104363-Karlsson1" target="_blank">[6]</a>.</p>1<p>Chromosome 20, Broad CanFam3.1, Sept 2011.</p>2<p>According to Karlsson <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104363#pone.0104363-Karlsson1" target="_blank">[6]</a>.</p

Allele frequencies of three candidate <i>MITF</i> polymorphisms in wolves from different geographic regions.

1<p>The frequency of the allele with the SINE insertion is presented.</p>2<p>The <i>A</i> and <i>G</i> alleles at SNP#21 are associated with white spotting and solid colour, respectively.</p

Overview of the different phenotypes included in the study.

<p>(A) Solid (<i>S/S</i>) Bull terrier, (B) white Bull terrier (<i>s^w</i>/<i>s^w</i>), (C) flash (<i>S/s^w</i>) Bull terrier, (D) Irish spotting (<i>sⁱ</i>/<i>sⁱ</i>) in a Bernese Mountain Dog, (E) piebald (<i>s^p</i>/<i>s^p</i>) Beagle, (F) wolf. Drawings: Anders Sundström.</p

A novel locus on chromosome 14 is associated with Shar-Pei amyloidosis. A

<p>. The genome wide association study of amyloidosis positive (<i>n</i> = 37) and negative (<i>n</i> = 14) individuals revealed a signal of association on chromosome 14 (14∶54948811, <i>p_MM</i> = 4.34×10⁻⁶) which was of slightly reduced strength to that observed on chromosome 13 (13∶27371905, <i>p_MM</i> = 3.0×10⁻⁶). <b>B</b>. A zoomed view of a subset of the genes across the chromosome 14 disease associated region, <b>C</b>. This region encompasses two peaks in moderate LD (<i>r²</i> = 0.6) as indicated by heat colouring. A genome wide significant threshold (red dotted line) has been defined using as Bonferroni 5%. <b>D</b>. The scaled plots of genes demonstrating differential expression between amyloidosis negative (<i>n</i> = 7) and positive (<i>n</i> = 7) kidney biopsies. Samples are shown in the same order for each gene and also in order of phased H14 haplotypes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075242#pone.0075242.s005" target="_blank">Table S3</a>). Each sample registered expression, although in some cases it was negligible. p-values 0.05>0.01, *>**.</p

The five investigated signs of Shar-Pei autoinflammatory disease share overlapping association signals.

<p>The phenotypes of fever (<b>A</b>. <i>n</i> = 129), arthritis (<b>B</b>. <i>n</i> = 107), vesicular hyaluronosis (<b>C</b>. <i>n</i> = 46), and otitis (<b>D</b>. <i>n</i> = 27) were compared to a subset of healthy controls at least 7 years old (<i>n</i> = 24). The phenotype of amyloidosis (<b>E</b>) required individuals to undergo renal biopsy screening to be declared positive (<i>n</i> = 37) or negative (<i>n</i> = 14) for the deposits. In each panel the top genome-wide significant SNP has been highlighted with an open circle and linkage disequilibrium (<i>r²</i>) between this marker and the others in the region coloured according to strength. A genome wide significant threshold (red dotted line) has been defined using an analysis specific Bonferroni 5% limit.</p

Summary of the separate GWAS analyses conducted on breed-subtype and five symptoms of Shar-Pei autoinflammatory disease (SPAID).

1<p>Number of markers from a genotyped set of 173,662 which passed two rounds of quality control.</p>2<p>The genomic inflation factor measured from the unadjusted qtscore GWAS.</p>3<p>The genomic inflation factor after the application of a polygenic mixed model encompassing the Identity By State (IBS) matrix.</p>4<p>Genomic position (<i>CanFam</i> 2.0, chromosome 13, bp) of the top SNP as ranked by ⁵Significance of mixed model p-value.</p>6<p>Number of SNP which exceeded the Bonferroni 5% threshold for significance.</p

Distribution of chromosome 14 Amyloidosis haplotype pairs in the total population of Shar-Pei analysed.

1<p>Phased haplotypes are composed of the top eight SNP identified from the chromosome 14 and are estimated from the total population frequency (<i>n = </i>255).</p>2<p>Pairs observed at frequencies less than 10% in a sample set have been collapsed.</p

Major haplotype pairs observed across chromosome 13 as defined by GWAS significant SNP.

1<p>Phased haplotypes are composed of the top SNP identified in each GWAS where genome-wide significance was reached and estimated from the total population frequency.</p>2<p>Haplotype pairs observed at frequencies less than 10% in a sample set have been collapsed.</p

The genetic signature of within breed sub-type on chromosome 13.

<p>Individuals representing the extremes of physical appearance (Meatmouth, heavily wrinkled thickened skin, <i>n</i> = 123; Bonemouth, few wrinkles left into adulthood, <i>n</i> = 33) were compared. <b>A</b>. Pairwise <i>F</i>_ST was calculated between the two groups for 126,206 SNP and plotted in chromosomal order. Smoothed values in bins of 100 SNP are superimposed to better illustrate the peak of association (red line). <b>B</b>. A zoomed view of a subset of the genes from the chromosome region in relation to the peak of <i>F</i>_ST (based on most divergent SNP and a 5% threshold, red box). <b>C</b>. The plot of a mixed model association test for breed subtype. The top genome-wide significant SNP (13∶23180227, <i>p_MM</i> = 2.63×10⁻⁹) has been highlighted with an open circle and linkage disequilibrium (<i>r²</i>) between this marker and the others in the region are coloured according to strength. A genome wide significant threshold (red dotted line) has been defined using an analysis specific Bonferroni 5% limit.</p