GENETIC POLYMORPHISMS ON G-QUADRUPLEXES AS A CAUSE OF ONCOGENES TRANSCRIPTIONAL AND TRANSLATIONAL EXPRESSION VARIATIONS.

PIGA, E.J.; LORENZATTI, A.; BINOLFI, A.; CALCATERRA, N.B.; ARMAS, P.

Marienbad (Marianské Lázne)

Congreso; G4thering 2022. 8th International Meeting on Quadruplex Nucleic Acids.; 2022

G4thering 2022. 8th International Meeting on Quadruplex Nucleic Acids.

G-quadruplexes (G4) have been proposed as transcriptional and translational regulatory elements originally and majorly described for oncogenes. On the other hand, genomic scale association studies by massive DNA sequencing revealed that single nucleotide polymorphisms (SPNs) associated with human diseases are frequently found near transcription start sites, within proximal promoter regions (PPRs) and 5’ untranslated regions (5' UTRs). The goal of this work was to identify SNPs overlapped with G4 forming sequences (G4S) described as transcriptional or translational regulators of oncogenes, that may most affect G4 folding, hereafter called SNP-G4S. First we performed a bioinformatic analysis using Ensembl database to identify the SNP-G4Ss found in G4s described as transcriptional or translational regulators of several oncogenes. For each reference sequence we generated a collection of variable sequences containing each polymorphism and a mutant sequence with no G4S (unable to form G4). Then we used several G4 folding predictors in order to identify those SNP-G4Ss that may affect G4 folding or stability. Based on the results of this analysis, we chose some SNP-G4Ss for further in vitro analyses. Circular Dichroism (CD) spectra, qPCR stop assays, CD melting assays and 1D 1H NMR analyses indicated that some SNP-G4Ss cause quantitative or qualitative spectral changes probably related with G4 stability changes. Finally, SNP-G4Ss that produced significant structural variations in vitro were cloned into pGL3 promoter vector and transfected into HEK293 cells, revealing that SNPs altered luciferase reporter activity. Results gathered in this work suggest that SNP-G4Ss that alter G4 folding may be the cause of differential expression of oncogenes and should be considered as a novel molecular etiology mechanism for the predisposition or establishment of diseases.