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Functional analysis of polymorphic genes enrolled in melanogenesis in cutaneous melanoma

Grant number: 16/02193-9
Support type:Regular Research Grants
Duration: November 01, 2016 - October 31, 2018
Field of knowledge:Biological Sciences - Genetics - Human and Medical Genetics
Principal Investigator:Gustavo Jacob Lourenço
Grantee:Gustavo Jacob Lourenço
Home Institution: Faculdade de Ciências Médicas (FCM). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil
Assoc. researchers:Carmen Silvia Passos Lima ; Manoela Marques Ortega


We recently observed for the first time 12,882 new single nucleotide polymorphisms (SNPs) in association with cutaneous melanoma (CM) risk in 103 samples of CM patients and 103 controls, using large-scale genotyping with DNA microarrays (SNP array 6.0, Affytmetrix®). Three new SNPs located in the regulatory sequence of RNA messenger processing (splicing) of genes related with melanogenesis, ADCY3 c.675+9196T>G (rs11900505), CREB1 c.303+373G>A (rs10932201) e MITF c.938-325A>G (rs7623610) were selected. The quantities and the functions of the proteins coded by wild-type and variant alleles of SNPs are unknown. Thus, the objectives of this study are verify whether the distinct genotypes of referred SNPs influence: 1) the MC risk, the clinical and tumor characteristics, 2) the progression-free survival and overall survival of MC patients; 3) the ADCY3, CREB1 and MITF (melanogenesis pathway), SF1 and HNRNPA1 (splicing mechanism) genes and protein expressions; and 4) the efficacy of the splicing mechanism of ADCY3, CREB1 and MITF genes by minigene reporter assays using melanoma cell line (A-375). In order to achieve the objectives, genomic DNA of peripheral blood of 250 MC patients and 250 controls will be evaluated to identify the genotypes of each SNP by real-time polymerase chain reaction (PCR), using TaqMan® assays. The expressions of genes and proteins will be evaluated in total RNA and proteins of peripheral blood from controls (15 with wild-type homozygous genotype, 15 heterozygous and 15 variant homozygous genotype of each SNP) by quantitative PCR, using specific primers and SYBR green dye, and western blotting method, respectively. The efficacy of splicing mechanism will be evaluated by minigene reporter assay. The differences between groups will be analyzed by Fisher and qui-square tests; and multiple logistic regression. The survival times will be analyzed by Kaplan-Meier method and, uni and multivariate Cox analyses. The differences between genes expressions will be evaluated by t test and ANOVA or Mann-Whitney and Kruskal-Wallis tests. The efficiency of splicing mechanism will be evaluated by analysis of the size of DNA fragments generated by distinct minigenes. We believe that results of this study will contribute to define the roles of the SNPs in the protein production and their roles in the MC inherited predisposition. (AU)

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