New susceptibility loci for cutaneous melanoma risk and progression revealed using a porcine model.

Despite major advances, it is estimated that a large part of melanoma predisposing genes remains to be discovered. Animal models of spontaneous diseases are valuable tools and experimental crosses can be used to identify and fine-map new susceptibility loci associated with melanoma. We performed a Genome-Wide Association Study (GWAS) of melanoma occurrence and progression (clinical ulceration and presence of metastasis) in a porcine model of spontaneous melanoma, the MeLiM pig. Five loci on chromosomes 2, 5, 7, 8 and 16 showed genome-wide significant associations (p < 5 × 10-6) with either one of these phenotypes. Suggestive associations (p < 5 × 10-5) were also found at 16 additional loci. Moreover, comparison of the porcine results to those reported by human melanoma GWAS indicated shared association signals notably at CDKAL1 and TERT loci but also nearby CCND1, FTO, PLA2G6 and TMEM38B-RAD23B loci. Extensive search of the literature revealed a potential key role of genes at the identified porcine loci in tumor invasion (DST, PLEKHA5, CBY1, LIMK2 and ETV5) and immune response modulation (ETV5, HERC3 and DICER1) of the progression phenotypes. These biological processes are consistent with the clinico-pathological features of MeLiM tumors and can open new routes for future melanoma research in humans.

Impact of a CD4 gene haplotype on the immune response in minipigs.

The cluster of differentiation 4 (CD4) molecule functions as a co-receptor for MHC class II binding to TCR in T helper cells. A CD4 epitope deficiency was identified in the swine MeLiM (melanoblastoma-bearing Libechov minipig) strain, a model for spontaneous cutaneous melanoma development and regression. Extensive sequencing revealed a high genetic variability of CD4 and the existence of several haplotypes segregating in MeLiM. Forty polymorphisms were identified in the coding sequence, out of which 20 correspond to non-synonymous variants and 10 are located in the 3'UTR of CD4 transcripts. One of the haplotypes segregating in the MeLiM explained the epitope deficiency observed. An association analysis between CD4 genotype and several phenotypes related to tumor regression was performed in 267 animals. An association was evidenced between a MeLiM alternative CD4 haplotype and skin and eye depigmentation, as well as the extent of hair depigmentation. Also, seric IgG concentration was shown to be higher in pigs carrying the alternative haplotype at the homozygous state. In conclusion, the genetic variability of the CD4 gene is associated with immune response-related phenotypes in MeLiM minipigs.

Genetic and functional evaluation of MITF as a candidate gene for cutaneous melanoma predisposition in pigs.

Cutaneous melanoma arises from transformed melanocytes and is caused mainly by environmental effects such as ultraviolet radiation and to a lesser extent by predisposing genetic variants. Only a few susceptibility genes for cutaneous melanoma have been identified so far in human; therefore, animal models represent a valuable alternative for genetic studies of this disease. In a previous quantitative trait locus (QTL) study, several susceptibility regions were identified in a swine biomedical model, the MeLiM (Melanoblastoma-bearing Libechov minipig) pigs. This article details the fine-mapping of a QTL located on SSC13 (Sus scrofa chromosome 13) through an increase in marker density. New microsatellites were used to confirm the results of the first analysis, and MITF (microphthalmia-associated transcription factor) was selected as a candidate gene for melanoma development. A single-marker association analysis was performed with single-nucleotide polymorphisms (SNPs) spread over the locus, but it did not reveal a significant association with diverse melanoma-related traits. In parallel, MITF alternative transcripts were characterized and their expression was investigated in different porcine tissues. The obtained results showed a complex transcriptional regulation concordant with the one present in other mammals. Notably, the ratio between MITF+ and MITF- isoforms in melanoma samples followed the same pattern as in human tumors, which highlights the adequacy of the MeLiM pig as a model for human melanoma. In conclusion, although MITF does not seem to be the causal gene of the QTL initially observed, we do not exclude a prominent role of its transcription and function in the outbreak and evolution of the tumors observed in pigs.

Detection of novel quantitative trait loci for cutaneous melanoma by genome-wide scan in the MeLiM swine model.

Human cutaneous melanoma is a complex trait inherited in about 10% of cases. Although 2 high-risk genes, CDKN2A and CDK4, and 1 low risk gene, MC1R, have been identified, susceptibility genes remain to be discovered. Here, we attempted to determine new genomic regions linked to melanoma using the pig MeLiM strain, which develops hereditary cutaneous melanomas. We applied quantitative trait loci (QTL) mapping method to a significant genome-wide scan performed on 331 backcross pigs derived from this strain. QTLs were detected at chromosome-wide level for a melanoma synthetic trait corresponding to the development of melanoma. The peak positions on Sus scrofa chromosomes (SSC) were at 49.4 and 88.0 cM (SSC1), 56.0 cM (SSC13), 86.5 cM (SSC15) and 39.8 cM (SSC17), and, on SSC2, at 16.9 cM, in families derived from F1 males only (p < 0.05, except for SSC13, p < 0.01). Analysis of 7 precise specific traits revealed highly significant QTLs on SSC10 (ulceration), on SSC12 (presence of melanoma at birth), on SSC13 (lesion type), and on SSC16 and SSC17 (number of aggressive melanomas) at the respective positions 42.0, 95.6, 81.0, 45.3 and 44.8 cM (p < 0.001 and p < 0.05 respectively at the chromosome- and genome-wide levels). We also showed that MeLiM MC1R*2 allele, which determines black coat colour in pigs, predisposes significantly to melanoma. Interactions were observed between MC1R and markers located on SSC1 (p < 0.05). Taken together, these results indicate that MeLiM swine is a model for human multigenic diseases. Comparative mapping revealed human regions of interest to search for new melanoma susceptibility candidates.

Identification of five chromosomal regions involved in predisposition to melanoma by genome-wide scan in the MeLiM swine model.

In human familial melanoma, 3 risk susceptibility genes are already known, CDKN2A, CDK4 and MC1R. However, various observations suggest that other melanoma susceptibility genes have not yet been identified. To search for new susceptibility loci, we used the MeLiM swine as an animal model of hereditary melanoma to perform a genome scan for linkage to melanoma. Founders of the affected MeLiM stock were crossed with each other and with healthy Duroc pigs, generating MeLiM, F1 and backcross families. As we had previously excluded the MeLiM CDKN2A gene, we paid special attention to CDK4 and MC1R, as well as to other candidates such as BRAF and the SLA complex, mapping them on the swine radiation hybrid map and/or isolating close microsatellite markers to introduce them into the genome scan. The results revealed, first, that swine melanoma was inherited as an autosomal dominant trait with incomplete penetrance, preferably in black animals. Second, 4 chromosomal regions potentially involved in melanoma susceptibility were identified on Sus Scrofa chromosomes (SSC) 1, 2, 7 and 8, respectively, in intervals 44-103, 1.9-18, 59-73 and 47-62 cM. A fifth region close to MC1R was revealed on SSC 6 by analyzing an individual marker located at position 7.5 cM. Lastly, CDK4 and BRAF were unlikely to be melanoma susceptibility genes in the MeLiM swine model. The 3 regions on SSC 1, 6 and 7, respectively, have counterparts on human chromosomes (HSA) 9p, 16q and 6p, harboring melanoma candidate loci. The 2 others, on SSC 2 and 8, have counterparts on HSA 11 and 4, which might therefore be of interest for human studies.

CDKN2A region polymorphism and genetic susceptibility to melanoma in the melim swine model of familial melanoma.

Some herds of miniature swine are genetically predisposed to cutaneous melanoma. To test if swine melanoma susceptibility could be linked to the CDKN2A gene, which is involved in a proportion of 9p21-linked human familial melanoma, we performed a genetic analysis of miniature pigs of the MeLiM strain. F(1) and backcross animals were generated by crossing 1 MeLiM boar with healthy Duroc sows. We isolated the swine CDKN2A gene and characterized a linked informative microsatellite marker, the S0644 marker. Using this marker and 2 flanking markers, we analyzed the segregation of the CDKN2A gene in a 3-generation pedigree. Allelic association, linkage analysis and haplotype analysis of these data led to exclusion of the CDKN2A gene as a candidate for melanoma susceptibility. Nonetheless, this analysis suggests an association with the swine 1q25 chromosomal region, which is homologous to the human 9p21 region.