Identification of mutations in canine oral mucosal melanomas by exome sequencing and comparison with human melanomas

Abstract

Oral mucosal melanomas (OMMs) are aggressive neoplasms commonly found in dogs but rare in humans. Utilizing whole exome sequencing (WES), which focuses on protein-coding regions to reveal mutation profiles, we conducted a comparative analysis of canine OMM and human melanomas. This study involved DNA extraction, exome enrichment, and sequencing from three canine OMM cell lines (CMGD-2, CMGD-5, TLM-1), five canine OMM frozen samples, a human OMM cell line (MEMO), and a human commercial skin melanoma cell line (SK-MEL-28). The sequencing and subsequent analysis of FASTQ files yielded final variant files, leading to the identification of mutations. Our findings revealed a total of 500 mutated genes in canine OMM, including significant ones such as EP300, FAT4, JAK3, LRP1B, NCOR1, and NOTCH1. Notably, 82 shared mutations were identified between human melanomas and canine OMM genomes. These mutations were categorized based on the gene functions. The identification of these mutations provides critical insights that can pave the way for the development of novel therapeutic strategies for both canine and human OMM, offering hope for more effective treatments in the future.

Keywords: Exome sequencing; Melanoma; Mucosa; Mutations; Variants.

Fig. 1

Pipelines used for the preparation of human (a) and canine (b) OMM samples for exome sequencing analysis and identification of mutations. Human melanoma cell lines (MEMO and SK-MEL-128) sequencings were compared to the reference genome hg38, while the canine OMM samples and cell lines sequencings were compared with the CanFam3 reference genome. Details of programs used for the analysis can be found in the text.

Fig. 2

(a) Distribution of different classes of variants among canine oral melanoma lineages; (b) Distribution of single nucleotide substitutions. The graph shows the prevalence of the six most common types of base exchanges in various strains of canine oral melanomas, encompassing high- and moderate-impact variants; (c) Distribution of different variants in protein-coding regions according to canine oral melanoma samples.

Fig. 3

Distribution of different classes of variants among human melanoma cell lines, MEMO and SK-MEL-28.

Fig. 4

Distribution of single nucleotide substitutions. The graph shows the distribution of the six most prevalent types of base exchanges in different human melanoma lineages.

Fig. 5

Distribution of different variants in protein-coding regions according to human melanoma samples.

Fig. 6

Oncoplot of cancer-related variants in canine OMM, including CMGD2m CMGD5, TLM1, M5, M12, M16, M38 and M41, showing the 50 most prevalent genes with changes.

Fig. 7

Mutations among genes with homologs in both humans and dogs that exhibited mutations in at least one of the samples. Notably, the CMGD-5 sample showed the highest level of mutations than the other samples. In contrast, the MEMO, a human oral melanoma line, presented the lowest level of mutations. MEMO and SK- MEL-28 are human melanoma cell lines (in blue); TLM-1, CMGD-2 and CMGD-5 are canine oral melanoma cell lines, and M16, M5, M41, M12 and M38 are canine oral melanoma frozen samples (in green).