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Functional analysis of "MITF" c.938-325G>A polymorphism, enrolled in melanogenesis, in cutaneous melanoma

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Caroline Torricelli
Total Authors: 1
Document type: Master's Dissertation
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Our group of researchers identified thousands of single-nucleotide gene variants (SNVs) associated with altered risk of cutaneous melanoma (MC) through large-scale genotyping with DNA microarray (SNV array 6.0, Affymetrix®). The SNV MITF rs7623610 (c.938-325G>A) stood out among them, because it is located in the splicing regulatory region of MITF gene, an important regulator of the melanogenesis pathway. Thus, the objectives of the study were to evaluate if the different genotypes of the referred SNV influence the risk of CM occurrence in a sample of adequate size, clinical and tumor aspects, the prognosis of patients, gene and protein expression of MITF and of SF1 and HNRNPA1 (splicing regulatory proteins) and the efficiency of the splicing mechanism. The genotypes of 247 MC patients and 280 controls were identified by real-time polymerase chain reaction (PCR). The RNA and protein samples from peripheral blood leukocytes from 41 patients and 83 controls were used to evaluate the expression of MITF, SF1 and HNRNPA1 by quantitative PCR and the protein content of controls by the western blotting technique. The splicing mechanism efficiency was evaluated by the minigene reporter assay in melanoma cell line. The differences between groups were assessed by multiple logistic regression, t-test, variance analysis, Mann-Whitney and Kruskal Wallis. The progression-free survival (SLP) and melanoma-specific survival (MSS) times were estimated using Kaplan-Meier and Cox methods. The frequency of MITF AA genotype was higher in patients than in controls (27.9% vs. 20.7%, p= 0.02). Individuals with the referred genotype were at about twice as likely the risk of developing MC than the others. MITF AA genotype was more common in patients with advanced stage tumors than the others (46.2% vs. 24.9%, p= 0.007). MSS was lower in patients with MITF AA genotype (77.1% vs. 90.1%, p= 0.03) and with GA or AA genotype (79.4% vs. 90.1%, p= 0.04). Patients or controls with MITF AA genotype presented lower expression of HNRNPA1 than those with the other genotypes (0.32 arbitrary units (AUs) vs. 0.88 AUs; p= 0.009 and 1.07 AUs vs. 1.30 AUs, p= 0.03, respectively). Melanoma cells carrying the minigene with the A allele of the referred SNV had lower expression of the MITF minigene. Our results suggest that SNV MITF rs7623610 is an important inherited factor for MC risk and prognosis (AU)

FAPESP's process: 16/02193-9 - Functional analysis of polymorphic genes enrolled in melanogenesis in cutaneous melanoma
Grantee:Gustavo Jacob Lourenço
Support type: Regular Research Grants