Human disease-related G4-Vars ? genetic variations can affect G-quadruplex formation and function

GISMONDI, MAURO; PIGA, ERNESTO; ARMAS, PABLO; MARGARIT, EZEQUIEL; BINOLFI, ANDRÉS; LORENZATTI, AGUSTÍN; CALCATERRA, NORA

Conferencia Virtual

Simposio; 5to SAJIB; 2020

RSG-Argentina

Human variation databases contain a vast amount of genetic variationsincluding single nucleotide polymorphisms (SNPs) that can show moleculareffects on coding and non-coding regions. Although many variations wereassociated with different human diseases by DNA sequencing studies from patientpopulations (Genome Wide Association Studies or GWAS), the molecular mechanismsfor most of them are unknown. G-quadruplexes (G4) are one of few secondarystructures on nucleic acids that can be transiently folded when single-strandedG-rich regions are exposed, and were involved in different nucleic acidsprocesses, including transcriptional gene expression regulation. The main goalof this work was to identify human diseases-related G4-Vars: naturally-ocurringvariations associated with human diseases whose versions possess contrastingand drastic abilities to affect G4 structure formation. First, 417071 sequencesflanking variations present in proximal promoter regions (PPRs - 1000 bpupstream from transcription start sites) and their corresponding polymorphisminformation were downloaded from COSMIC, ClinVar, dbSNP and HGMD-PUBLIC humanvariation databases. We substituted the polymorphism position with reference oralternative versions. A G4 lineal pattern analysis showed that about 3% of thetotal sequences possess a G4 motif in one or more of their associatedsubstituted sequences. Comparing reference and alternative versions for eachsequence led us to get  2312 G4-Varsequences whose versions allowed  or avoided  G4 motif occurrence.The implementation of a novel G4 folding predictor (QUADRON) with a machinelearning model based on in vitro G4genomic data, supported 657 G4-Vars to have the best chances to differentiallyoccur due to polymorphisms in genomic sequences. Following in silico predictions, disease-related G4-Vars on the PPRs of GRIN2B, F7, CSF2 and SIRT1 genes were experimentallyvalidated by in vitro analyses andtranscriptional regulation assays using reporter genes in cultured cells. Results suggestG4-Vars as a novel mechanism affecting differential transcription and pointthat they should be considered to understand the molecular effect of variationson the predisposition or establishment of human diseases and as potentialtargets for chemotherapeutic treatments.