GENETIC POLYMORPHISMS IN G-QUADRUPLEX AFFECT THE TRANSCRIPTION OF HUMAN DISEASE-RELATED GENES

LORENZATI, A. ; GISMONDI, M.; ARMAS, P.; MARGARIT, E.; CALCATERRA, N.B.; PIGA, E.; BINOLFI, A.

Salta

Congreso; Joint LV Annual SAIB Meeting and XIV PABMB Congress; 2019

SAIB and PABMB

G-quadruplexes (G4) are secondary structures that can be transiently folded within proximal promoter regions (PPRs) during transcription when single-stranded G-rich regions are exposed. Genomic scale association studies by massive DNA sequencing revealed that single nucleotide polymorphisms (SPNs) associated with human diseases are mainly present nearby transcription start sites (TSS) within promoters and 5´ untranslated regions. The goal of this work was to identify SNPs associated with human diseases that are located in PPRs (here defined as the -1000 pb from TSS) and may affect the folding of putative G4 (PG4), hereafter called SNP-PG4. First, we performed a bioinformatic analysis by downloading (using Ensembl Biomart tool) the flanking sequences (+/- 50 bp) of each SNP located within the PPR associated with human diseases from COSMIC, ClinVar, dbSNP and HGMD. Nearly 3% of the 427479 sequences with at least two variants contained PG4s. To identify those SNPs most affecting G4 folding we selected sequences containing at least one PG4 in either the reference or the variant versions; i.e., SNPs that disrupt or promote the G4-folding. Then we used a novel G4 folding predictor based on a large-scale machine learning from an extensive experimental G4-formation dataset (Quadron) to choose for those PG4s with the higher scores. SNP-PG4 within the PPRs of the genes GRIN2B, F7, CSF2 and SIRT1 were further analyzed. Spectroscopic analyses by Circular Dichroism (CD) demonstrated that the of the SNP-PG4 identified GRIN2B and F7 fold in vitro as G4 and that SNPs cause quantitative spectral changes. Moreover, 1D 1H NMR spectroscopy confirmed the formation of the G4s identified for GRIN2B and F7 and that SNPs induce quantitative and qualitative changes. qPCR stop assays and CD melting assays indicated that SNPs induce G4 stability changes. Finally, PG4s cloning into pGL3 promoter vector revealed that firefly luciferase reporter activity was altered by SNPs when transfected into HEK293 cells. GRIN2B codes for a subunit of glutamate receptor and has been reported related to Parkinson?s, Huntington?s, and Alzheimer?s diseases and a broad spectrum of neurodevelopmental disorders. F7 codes for coagulation factor VII, a member of the coagulation cascade which miss-regulation causes rare severe bleeding disorders known as congenital factor VII deficiency. Results gathered in this work suggest that SNPs in the PPRs of these genes may alter G4 folding thus modifying transcriptional activity. In view of this, SNP-PG4s should be considered as a novel molecular etiology mechanism for the predisposition or establishment of human diseases, as well as potential targets for chemotherapeutic treatments.