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

Comparison of canFam2.0 and canFam3.1.

<p>Comparison of canFam2.0 and canFam3.1.</p

Distance trees of expression profiles.

<p>We constructed neighbor-joining trees based on the correlation between expression values (FPKM>1.0) between samples, with 1 minus Spearman's rho defining the distance. Colors denote library construction methods (poly-A: blue, DSN: red). We divided transcribed loci into (a) protein coding genes with RNA-Seq support, either annotated by EnsEMBL in dog or EnsEMBL in the human orthologous regions. Replicates cluster together, so do the library constructions methods poly-A and DSN, as well as related tissues, such as heart and muscle; (b) antisense transcripts, that overlap at least one exon of a protein coding gene, as defined in (a). With the exception of testis, poly-A and DSN separate the samples, with both the poly-A and DSN sub-trees maintaining closer relationships between the related tissues heart and muscle; (c) spliced intergenic loci, excluding sequences that have coding potential. Similar to protein coding genes, the poly-A and DSN group by tissue first, with the exception of kidney DSN; and (d) intergenic and uncharacterized single-exon transcript loci. In this set, DSN and poly-A are, similar to antisense loci, the most dominant factor when grouping samples.</p

Location of lincRNAs and single-exon intergenic non-coding transcripts.

<p>(a) We show the mapping of lincRNAs, broken down by sample across chromosome 1. (b) Histogram of distance from intergenic transcripts to the next known transcribed element in both the 5′ (left) and 3′ (right) direction. The average distance is around 150,000 nucleotides, suggesting that these loci are not closely associated with known genes.</p

Transcribed loci per tissue and library preparation.

<p>N/A, due to poor alignment performance, this library was excluded from subsequent analyses.</p