A synonymous germline variant in a gene encoding a cell adhesion molecule is associated with cutaneous mast cell tumour development in Labrador and Golden Retrievers

Abstract

Mast cell tumours are the most common type of skin cancer in dogs, representing a significant concern in canine health. The molecular pathogenesis is largely unknown, but breed-predisposition for mast cell tumour development suggests the involvement of inherited genetic risk factors in some breeds. In this study, we aimed to identify germline risk factors associated with the development of mast cell tumours in Labrador Retrievers, a breed with an elevated risk of mast cell tumour development. Using a methodological approach that combined a genome-wide association study, targeted next generation sequencing, and TaqMan genotyping, we identified a synonymous variant in the DSCAM gene on canine chromosome 31 that is associated with mast cell tumours in Labrador Retrievers. DSCAM encodes a cell-adhesion molecule. We showed that the variant has no effect on the DSCAM mRNA level but is associated with a significant reduction in the level of the DSCAM protein, suggesting that the variant affects the dynamics of DSCAM mRNA translation. Furthermore, we showed that the variant is also associated with mast cell tumours in Golden Retrievers, a breed that is closely related to Labrador Retrievers and that also has a predilection for mast cell tumour development. The variant is common in both Labradors and Golden Retrievers and consequently is likely to be a significant genetic contributor to the increased susceptibility of both breeds to develop mast cell tumours. The results presented here not only represent an important contribution to the understanding of mast cell tumour development in dogs, as they highlight the role of cell adhesion in mast cell tumour tumourigenesis, but they also emphasise the potential importance of the effects of synonymous variants in complex diseases such as cancer.

Fig 1. GWAS meta-analysis of MCT in Labrador Retrievers.

A. Manhattan plot of the combined analysis of 105 cases and 85 controls from three case-control sets (Sets 1–3). Analyses comprised 115,432 SNPs. B. Regional association plot highlighting the regions surrounding the signal for MCT in Labrador Retrievers. The horizontal red line denotes the genome-wide association threshold based on Bonferroni correction for 115,432 tests (P-value = 4.3 x 10⁻⁷). The horizontal blue line represents the empirical statistical threshold used to delineate the critical region surrounding the top SNP (P-value<0.01). Plots were generated using Haploview version 4.2 [74].

Fig 2. SNP rs850678541 genotyping of Labrador Retriever tissue biopsies.

A. Allelic discrimination plot, generated by the TaqMan Genotyper Software, showing the distribution of the 20 biopsies analysed. Reference allele: G, and alternative allele: A. The SNP rs850678541 genotypes are represented by: blue spheres—Alternative (variant) ‘A’ allele homozygote; red spheres—Reference ‘G’ allele homozygote; green spheres—G/A heterozygote. B. Table showing the status and SNP rs850678541 genotype of each biopsy.

Fig 3. DSCAM mRNA levels in MCTs from Labrador Retrievers with different SNP rs850678541 genotypes.

A. Bar charts showing the DSCAM mRNA level (anti-log of the CNRQ—Calibrated Normalised Relative Quantity—values) of the indicated biopsies, grouped by their SNP rs850678541 genotype. The differences between the groups (as assessed by Kruskal-Wallis and Mann-Whitney U tests, respectively) were not statistically significant. B. Bar charts showing the mean DSCAM mRNA level (anti-log of the CNRQ values) for each genotype group. Error bars represent standard deviations. The SNP rs850678541 genotypes are represented by: ALT/ALT—Alternative (variant) ‘A’ allele homozygote; REF/REF—Reference ‘G’ allele homozygote; REF/ALT—GA heterozygote.

Fig 4. DSCAM protein levels in MCTs from Labrador Retrievers with different SNP rs850678541 genotypes.

A. DSCAM western blot prepared using protein samples extracted from the indicated MCT biopsies. Ponceau S total protein staining was used for normalisation to adjust for variation in protein loading. Sample number colours indicate the SNP rs850678541 genotype: Red = Alt/Alt [Alternative (variant) ‘A’ allele homozygote]; Green = Ref/Alt [GA heterozygote]; Blue = Ref/Ref [Reference ‘G’ allele homozygote]. B. Bar charts showing the DSCAM protein levels of the indicated biopsies, grouped by their SNP rs850678541 genotype, as quantified by the Image Lab software (using Ponceau S-measured total protein quantity as the normaliser, and ‘Sample 6’ as the inter-membrane calibrator). *Mann-Whitney two-tailed U test P = 0.04. C. Bar charts showing the mean DSCAM protein level of each SNP rs850678541 genotype group. Error bars represent standard deviations.

Fig 5

DSCAM protein levels in normal skin biopsies from Labrador Retrievers with different SNP rs850678541 genotypes: A. DSCAM protein levels in normal skin biopsies from Labrador Retrievers with different SNP rs850678541 genotypes: A. DSCAM Western blot prepared using protein samples extracted from the indicated skin biopsies (Fig 2B IDs: 18, 19, 20). Ponceau S total protein staining was used for normalisation to adjust for variation in protein loading. Sample number colours indicate the SNP rs850678541 genotype: Red = Alt/Alt [Alternative (variant) ‘A’ allele homozygote]; Blue = Ref/Ref [Reference ‘G’ allele homozygote]. B. Histogram showing the DSCAM protein levels of the indicated biopsies, as quantified by the Image Lab software (using Ponceau S-measured total protein quantity as the normaliser, and ‘Sample 6’ as the inter-membrane calibrator).

Fig 6

Assay for DSCAM alternative splicing: A. PCR amplicons derived from 7,725b DSCAM transcript ENSCAFT00000016117 expected to be amplified by the E14-15 and E-15-17 Assays. The DSCAM exonic sequences illustrated are minus DNA strand sequences. B. Gel Image showing the PCR products obtained by running the indicated PCR assays on MCT cDNAs. The sample numbers represent the IDs. of the MCT biopsies from which RNA was isolated. Sample number colours indicate the SNP rs850678541 genotype: Red = Alt/Alt [Alternative (variant) ‘A’ allele homozygote]; Blue = Ref/Ref [Reference ‘G’ allele homozygote].