Quantitative PCR results.

<p>The upregulation or downregulation of selected genes in STHS (Soft Tissue Histiocytic Sarcoma) and VHS (Visceral Histiocytic Sarcoma). The thick black line represents the median (50th percentile) and also the first and third quartile (25th and 75th percentile respectively) are displayed.Three genes are significantly differentially expressed: <i>C6</i> was up-regulated when comparing the more aggressive visceral histiocytic sarcoma to the soft tissue form, and <i>CLEC12A</i> and <i>CCL5</i> were down-regulated when comparing the more aggressive visceral histiocytic sarcoma to the soft tissue histiocytic sarcoma. Abbreviations<i>: C-type lectin domain family 12, member A (CLEC12A); C-C motif chemokine 5 Precursor (Small-inducible cytokine A5) (CCL5_CANFA); Asporin (ASPN); CD9 molecule (CD9);Transketolase-like 1, transcript variant 1 (TKTL1); Complement component 6, transcript variant 1(C6); S100 calcium binding protein A12 (S100A12); Immunoglobulin J polypeptide (IGJ); S100 calcium binding protein A8 (S100A8); Phytanoyl-CoA dioxygenase, peroxisomal like (PHYH).</i></p

Microarray- based heatmap of 11 genes.

<p><i>C-type lectin domain family 12, member A (CLEC12A); C-C motif chemokine 5 Precursor (Small-inducible cytokine A5) (CCL5_CANFA); Asporin (ASPN); CD9 molecule (CD9);Transketolase-like 1, transcript variant 1 (TKTL1); Complement component 6, transcript variant 1(C6); S100 calcium binding protein A12 (S100A12); Immunoglobulin J polypeptide (IGJ); S100 calcium binding protein A8 (S100A8); Phytanoyl-CoA dioxygenase, peroxisomal like (PHYH).</i></p

Additional file 3: of A nonsense mutation in B3GALNT2 is concordant with hydrocephalus in Friesian horses

Genes located in the ECA1 region shared in a homozygous state by hydrocephalus cases. Start and stop position (in base pair; Equus caballus EquCab2.0 reference genome [29]), symbol and description of genes located in the region of 1.47 Mb in length that is shared in a homozygous state by hydrocephalus cases. (DOCX 21 kb

QPCR primers for genes of interest (based on Microarray analyses) and reference genes (efficiencies varied between 95.0 and 104.8%).

<p>Genes identified using microarray as being significantly different comparing Soft Tissue Histiocytic Sarcoma (STHS) and Visceral Histiocytic Sarcoma (VHS): <i>C-type lectin domain family 12, member A (CLEC12A); C-C motif chemokine 5 Precursor (Small-inducible cytokine A5) (CCL5_CANFA); Asporin (ASPN); CD9 molecule (CD9);Transketolase-like 1, transcript variant 1 (TKTL1); Complement component 6, transcript variant 1(C6); S100 calcium binding protein A12 (S100A12); Immunoglobulin J polypeptide (IGJ); S100 calcium binding protein A8 (S100A8); Phytanoyl-CoA dioxygenase, peroxisomal like (PHYH)</i><u>Reference genes primers for q PCR:</u><i>Hypoxanthine phosphoribosyltransferase (HPRT), Ribosomal protein S19 (RPS19) ribosomalprotein L8 (RPL8), Signal recognition particle receptor (SRPR), Ribosomal protein L13, (RPL13), glucuronidase, beta (GUSB), Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH), Beta-2-Microglobulin (B2MG), 40S ribosomal protein S5 (RPS5), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-2-microglobulin (B2MG), Ribosomal protein S5 (RPS5).</i></p

Additional file 1: of A nonsense mutation in B3GALNT2 is concordant with hydrocephalus in Friesian horses

Sanger DNA sequencing results for the nonsense mutation. A table with information on all horses that have been sequenced for the nonsense mutation. (DOCX 20 kb

Altered Subcellular Localization of Heat Shock Protein 90 Is Associated with Impaired Expression of the Aryl Hydrocarbon Receptor Pathway in Dogs

<div><p>The aryl hydrocarbon receptor (AHR) mediates biological responses to toxic chemicals. An unexpected role for AHR in vascularization was suggested when mice lacking AHR displayed impaired closure of the ductus venosus after birth, as did knockout mice for aryl hydrocarbon receptor interacting protein (AIP) and aryl hydrocarbon receptor nuclear translocator (ARNT). The resulting intrahepatic portosystemic shunts (IHPSS) are frequently diagnosed in specific dog breeds, such as the Irish wolfhound. We compared the expression of components of the AHR pathway in healthy Irish wolfhounds and dogs with IHPSS. To this end, we analyzed the mRNA expression in the liver of <i>AHR</i>,<i>AIP</i>, <i>ARNT</i>, and other genes involved in this pathway, namely, those for aryl hydrocarbon receptor nuclear translocator 2 (<i>ARNT2</i>), hypoxia inducible factor 1alpha (<i>HIF1A</i>), heat shock protein 90AA1 (<i>HSP90AA1</i>), cytochromes P450 (<i>CYP1A1, CYP1A2</i>, and <i>CYP1B1</i>), vascular endothelial growth factor A (<i>VEGFA</i>), nitric oxide synthesase 3 (<i>NOS3</i>), and endothelin (<i>EDN1</i>). The observed low expression of <i>AHR</i> mRNA in the Irish wolfhounds is in associated with a LINE-1 insertion in intron 2, for which these dogs were homozygous. Down regulation in Irish wolfhounds was observed for <i>AIP</i>, <i>ARNT2</i>, <i>CYP1A2</i>, <i>CYP1B1</i> and <i>HSP90AA1</i> expression, whereas the expression of <i>HIF1A</i> was increased. Immunohistochemistry revealed lower levels of AHR, HIF1A, and VEGFA protein in the nucleus and lower levels of ARNT and HSP90AA1 protein in the cytoplasm of the liver cells of Irish wolfhounds. The impaired expression of HSP90AA1 could trigger the observed differences in mRNA and protein levels and therefore explain the link between two very different functions of AHR: regulation of the closure of the ductus venosus and the response to toxins.</p> </div

Comparison of <i>AHR</i> pathway gene expression in livers with a shunt and normal livers.

<p>The pattern of expression of selected genes in liver samples from Irish wolfhounds with an intrahepatic shunt (Iw), dogs with intrahepatic portosystemic shunts (IHPSS), and healthy dogs (H). The thick black line represents the median (50th percentile); the first and third quartile (25th and 75th percentile, respectively) are displayed. Outliers are depicted with an open dot, representing values higher than 1.5 times the interquartile range. HIF1A was found to be significantly upregulated in Irish wolfhounds with IHPSS, whereas <i>AHR</i>, <i>AIP</i>, <i>ARNT2, CYP1A2, CYP1B1, HSP90AA1, NOS3,</i> and <i>VEGFA</i> were significantly down regulated. No differences were observed in <i>ARNT</i>, <i>CYP1A1</i>, and <i>EDN1</i>. FC = fold change.</p

Significant immunohistochemical staining of liver tissue.

<p>Representative images of immunoreactivity in sections of formalin-fixed, paraffin-embedded liver samples from control dogs, dogs with extrahepatic and intrahepatic portosystemic shunts, and Irish Wolfhounds. Immunoreactivity against the aryl hydrocarbon receptor (AHR)(A), aryl hydrocarbon receptor nuclear translocator (ARNT)(B), hypoxia inducible factor 1alpha (HIF1A)(C), heat shock protein 90kDa alpha (cytosolic), class A member 1 (HSP90AA1)(D), and vascular endothelial growth factor A (VEGF)(E) is visible in variable intensity in the cytoplasm and/or nuclei of hepatocytes.</p

Immunohistochemistry of AHR pathway proteins in liver samples from healthy dogs, dogs with extrahepatic (EPHSS) or intrahepatic (IPHSS) portosystemic shunts, and Irish wolfhounds.

<p>The semi-quantative scoring in hepatocytes from Irish wolfhounds (Iw, n = 11), dogs with intrahepatic portosystemic shunts (IHPSS, n = 6), dogs with extrahepatic portosystemic shunts (EHPSS, n = 11) and healthy beagles (H, n = 6). The indicated p-values are obtained comparing Irish wolfhounds with the other groups as a whole.</p

LINE-1 insertion in intron 2 of <i>AHR</i> in Irish Wolfhounds.

<p>Southern blot analysis (A) with HindIII (lanes 1–4) and PstI (lanes 5–8) digested genomic DNA. Digestion with HindIII resulted in an increase in size of about 2000 bp in Irish wolfhounds (lanes 1 and 2) compared to healthy beagles (lanes 3 and 4). Using PstI as restriction enzyme results in a small increase in size of approximately 400 bp in Irish wolfhounds (lanes 5 and 6) compared to healthy beagles (lanes 7 and 8). The observed size differences are smaller than the actual rearrangement due to the presence of restriction enzyme sites in the rearranged DNA fragment. Gel electrophoresis of PCR products (B) with forward primer in exon 2 and reverse primer downstream intron 2. The use of Phusion ™ Hot Start High-Fidelity DNA Polymerase in combination with GC-buffer was required to amplify this fragment. An increase in intron size was detected in all used gDNA samples of Irish wolfhounds. Lane 1∶1 kb DNA ladder (Promega, Leiden, the Netherlands); 2: normal control; 3–5: genomic DNA of Irish wolfhounds; 6: negative control; 7∶100 bp DNA ladder (Promega). A 6298 bp long LINE-1 insertion named L1-Y_CF (C) is located 63 bp downstream of exon2. It contains a 13 bp long duplication site specific for LINE-1 insertions. In both open reading frames of this retrotransposon deletions were detected causing frame shifts and premature stop codons. Restriction sites were found for HindIII at 1908 bp and for PstI at 447 bp.</p