P-cadherin mutations are associated with high basal Wnt activity and stemness in canine mammary tumor cell lines

Purpose: To find underlying mutations causing highly-activated Wnt activity in mammary tumor cell lines associated with rounded morphology indicative of stemness/EMT. Methods: Stemness of high Wnt cell lines was confirmed using qPCR on selected genes and microRNA profiling, followed by whole-exome sequencing of 3 high Wnt canine mammary tumor cell lines and 5 low/absent Wnt cell lines. Candidate genes were identified and their involvement in Wnt activity investigated using siRNA silencing. Results: The high Wnt cell lines had morphological and gene expression characteristics reminiscent of stemness. All individual cell lines had about 4000 mutations in the exome in comparison to the reference canine genome. The three high basal Wnt cell lines had 167 unique exome mutations. Seven of these mutations resulted in a SIFT score <0.2 of proteins related to Wnt signaling. However, gene silencing did not change the Wnt pathway activation. Renewed analysis with respect to putative relations to Wnt signaling revealed that P-cadherin (CDH3) had three mutations in the coding region of the extracellular domain and was associated with high Wnt signaling. Silencing by siRNA not only in lowered Wnt activity, but also decreased levels of phosphorylated cSRC and sP-cad, and changed cell morphology towards spindle cell appearance. Conclusion: It is concluded that expression of mutated CDH3 is associated with activation of cSRC, stabilization of ß-catenin and a rounded morphology related to a stemness/EMT phenotype. A decreased Wnt activity can be found also by cSRC inhibition, but CDH3 silencing has an additional effect on morphology indicating reversal of EMT

P-cadherin mutations are associated with high basal Wnt activity and stemness in canine mammary tumor cell lines

Purpose: To find underlying mutations causing highly-activated Wnt activity in mammary tumor cell lines associated with rounded morphology indicative of stemness/EMT. Methods: Stemness of high Wnt cell lines was confirmed using qPCR on selected genes and microRNA profiling, followed by whole-exome sequencing of 3 high Wnt canine mammary tumor cell lines and 5 low/absent Wnt cell lines. Candidate genes were identified and their involvement in Wnt activity investigated using siRNA silencing. Results: The high Wnt cell lines had morphological and gene expression characteristics reminiscent of stemness. All individual cell lines had about 4000 mutations in the exome in comparison to the reference canine genome. The three high basal Wnt cell lines had 167 unique exome mutations. Seven of these mutations resulted in a SIFT score <0.2 of proteins related to Wnt signaling. However, gene silencing did not change the Wnt pathway activation. Renewed analysis with respect to putative relations to Wnt signaling revealed that P-cadherin (CDH3) had three mutations in the coding region of the extracellular domain and was associated with high Wnt signaling. Silencing by siRNA not only in lowered Wnt activity, but also decreased levels of phosphorylated cSRC and sP-cad, and changed cell morphology towards spindle cell appearance. Conclusion: It is concluded that expression of mutated CDH3 is associated with activation of cSRC, stabilization of ß-catenin and a rounded morphology related to a stemness/EMT phenotype. A decreased Wnt activity can be found also by cSRC inhibition, but CDH3 silencing has an additional effect on morphology indicating reversal of EMT

Whole genome homozygosity mapping results.

<p>A red peak reaching a 1.0 max score of statistical significance depicts a homozygous region on chromosome 19. The region coincides with the 0.49 Mbp locus identified using two-point linkage analysis and is bracketed by recombination spots (indicated in red). The identified haploblock fulfills the zygosity criterion by displaying homozygosity in affected animals (indicated in red) and heterozygosity in unaffected carriers (indicated in green).</p

Sanger sequencing confirmation of the disease segregating variant identified in the linked region.

<p>Candidate region identified using linkage analysis in PACG pedigree animals (left). Sequence chromatograms from a carrier (C), confirms heterozygous genotype whereas affected animals (A) display a homozygous state of variant identified in <i>NEB</i> (g.5588214 A->G) (Right).</p

A Fisher exact contingency table of genotypes observed in a confirmatory animal cohort for the <i>RIF1</i> variant (g.55723957 C->T).

<p>Forty-four additional unaffected and affected Basset Hounds were selected for confirmatory sequencing of the <i>RIF1</i> variant (g.55723957 C->T). The observed total of individuals and percentage of individuals displaying a specific genotype is shown for each cell respectively. The two-tailed P value is 0.57 (Fisher Exact Probability Test)</p><p>A Fisher exact contingency table of genotypes observed in a confirmatory animal cohort for the <i>RIF1</i> variant (g.55723957 C->T).</p

A Fisher exact contingency table of genotypes observed in a confirmatory animal cohort for the <i>NEB</i> variant (g.55885214 A->G).

<p>Forty-four additional unaffected and affected Basset Hounds were selected for confirmatory sequencing of the <i>NEB</i> variant (g.55885214 A->G). The observed total of individuals and percentage of individuals displaying a specific genotype is shown for each cell respectively.</p><p>The two-tailed P value is 0.00034 (Fisher Exact Probability Test)</p><p>A Fisher exact contingency table of genotypes observed in a confirmatory animal cohort for the <i>NEB</i> variant (g.55885214 A->G).</p

Alignment of the amino acid sequence of exon 48 in Nebulin in several vertebrate species.

<p>The Lysine (K) residue (highlighted in blue) at position p.2051 is conserved among 23 vertebrate species.</p

Basset Hound Pedigree used in this study.

<p>The affected Basset 5a in the second generation was duplicated twice (5b and 5c) in order to break two otherwise computationally confounding breeding loops. Genotypes of typed markers within region uncovered following two-point linkage analysis are shown. Shading indicates the transmission pattern of heterozygous parental haplotypes to affected, homozygous offspring. Additional patterns of shading including diagonal lines indicate variation to the same haplotype identified in the other pedigree members. Complete concordance of homozygous haplotype inheritance with the disease phenotype is observed in all affected animals.</p

Genome-wide linkage analysis of SNP genotype data.

<p>Using two-point linkage analysis, a maximum LOD_two-point score of 3.07 was achieved for a 0.49 Mb locus on chromosome 19. The red line of statistical significance indicates LOD scores values > 3 (A). Using multipoint linkage analysis, an increased maximum LOD_multipoint score of 3.24 was achieved for a locus mapped to the same location (B). A schematic view of the maximum LOD scores achieved across chromosome 19. The statistically significant locus is located at the distal end of chromosome 19 (Chr19: 54,949,124–56,765,346) and spans 1.82 Mbp (C).</p

Whole Exome Sequencing in Patients with the Cuticular Drusen Subtype of Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in elderly people worldwide. Cuticular drusen (CD) is a clinical subtype of AMD, which typically displays an earlier age at onset, and has a strong genetic component. Genetic studies support a role for rare sequence variants in CD susceptibility, and rare sequence variants in the CFH gene have been identified in 8.8% of CD cases. To further explore the role of rare variants in CD, we performed whole exome sequencing (WES) in 14 affected members of six families and 12 sporadic cases with CD. We detected rare sequence variants in CFH and FBLN5, which previously were shown to harbor rare variants in patients with CD. In addition, we detected heterozygous rare sequence variants in several genes encoding components of the extracellular matrix (ECM), including FBLN1, FBLN3/EFEMP1, FBLN5, FBLN6/HMCN1, FBN2, and COL15A1. Two rare pathogenic variants were identified in the COL15A1 gene: one in a sporadic case and another was found to segregate in a family with six affected individuals with CD. In addition, two rare pathogenic variants were identified in the FGL1 gene in three unrelated CD cases. These findings suggest that alterations in the ECM and in the coagulation pathway may play a role in the pathogenesis of CD. The identified candidate genes require further analyses in larger cohorts to confirm their role in the CD subtype of AMD. No evidence was found of rare sequence variants in a single gene that segregate with CD in the six families, suggesting that the disease is genetically heterogeneous