Molecular characterization of novel candidate genes involved in the initiation of multiple primary melanomas

Tese de mestrado, Biologia Molecular e Genética, Universidade de Lisboa, Faculdade de Ciências, 2019Cutaneous melanoma is considered one of the most aggressive human cancers due to its high invasive capacity. Several risk factors influence the development of melanoma, from which a genetic background is included. Approximately 5-12% of melanomas are familial cases, which comprise a higher frequency of multiple primary melanomas (MPMs). Approximately 20-40% of familial cases occur due to mutations in CDKN2A, a high-risk gene for cutaneous melanoma susceptibility. Other genes have also been described, although in a much lower percentage. Nevertheless, about 60-80% of all familial cases are caused by unknown genes. The aim of this study was the identification of novel susceptibility genes for MPMs. For this, whole exome sequencing was performed in 8 patients diagnosed with MPM, including two first-degree relatives, negative for germline mutations in the CDKN2A, CDK4 and MITF (p.E318K) genes. Afterwards, bioinformatics analysis, by the use of several criteria, allowed the identification of 19 variants potentially pathogenic, from which 5 were not validated as they were WES artefacts. Consequently, 13 variants were validated by the Sanger method: MAP2K3 (c.77G>C), MTCL1 (c.4315G>A), BMX (c.851C>T), NTN4 (c.1182C>T), FNDC1 (c.3332G>A), CDH23 (c.1096G>A), BRD9 (c.183G>C), CAND2 (c.992A>T), CFAP47 (c.4589A>C), ITIH3 (c.1130G>A), RNF213 (c.2122C>G), RPL32 (c.98G>A) and ARHGEF40 (c.2500C>T). Next, validated variants were screened, using the Sanger method, in 22 additional patients with MPM, 33 patients with familial melanoma and 100 healthy controls. Their association with MPM was calculated by the Fisher’s exact test, with an adjusted p-value using the BH method. In silico analysis was also performed for variant impact prediction using several tools, namely, SIFT, Polyphen-2, Provean, FATHMM, MutationTaster and Human Splicing Finder. A candidate gene literature search and gene analysis were also conducted for understanding the candidate genes function and possible relation to melanoma, respectively. Our results led to the identification of three potential mutations, specifically, CDH23 (c.1096G>A), BRD9 (c.183G>C) and ARHGEF40 (c.2500C>T), from which the BRD9 variant had a significant association with MPM. Furthermore, given their respective gene functions and prediction as damaging by several in silico tools, these appear to be promising pathogenic mutations. Several polymorphisms were also detected, therefore we hypothesized these might act together to increase melanoma susceptibility in a polygenic manner. However, functional studies should be conducted, to better understand the variants effect in normal and cancer cell lines and to evaluate their relevance in melanoma. In case of variant pathogenicity confirmation, the implementation of a NGS multigenic panel should also be considered in order to study the genes that harbor these variants as well as known susceptibility genes for melanoma