Identificação e caracterização funcional de variantes genéticas regulatórias da expressão gênica na hipercolesterolemia familiar

Abstract

Familial hypercholesterolemia (FH) is an inherited disorder of cholesterol metabolism, characterized by prolonged exposure to high serum concentrations of low-density lipoprotein (LDL) cholesterol, which leads to a significantly increased risk of early cardiovascular morbidity and death. The main causes of FH are variants found in genes involved in the LDL clearance pathway (LDLR, APOB and PCSK9), however, causal variants are not identified in a significant portion of clinically diagnosed individuals. Therefore, variants in regions that are not usually included in diagnostic panels, could be influencing the expression of these genes, and contributing to the phenotype. The present study aimed to identify and functionally characterize genetic variants located upstream of FH-related genes. 25 volunteers clinically diagnosed with FH but without confirmatory molecular diagnosis were selected for targeted sequencing of 3 kb upstream regions (Chr19:11086362-11089361; Chr2:21044074- 21047073; Chr1:55036476-55039475 - GRCh38) using the AmpliSeq for Illumina method and the Miseq Reagent Nano Kit v2. Computational analysis of sequencing results included pre-processing, mapping of the generated reads, variant calling, and visual inspection of alignments to confirm the quality of variant calling. Potentially regulatory variants were prioritized according to regulatory probability prediction algorithm, functional annotation with data from public databases, pathogenicity prediction algorithms, variant-related literature, and prediction of altered transcription factor binding. The effect of two of these potentially regulatory variants on the functioning of the promoters of their target genes was evaluated by luciferase reporter gene assays in HepG2 cells. 34 single nucleotide variants (SNV) identified in sequencing, passed the quality filters: 6 were upstream from LDLR, 15 from APOB and 13 from PCSK9. 5 variants were considered potentially regulatory and, consequently, good candidates for functional validation assays: rs36218923-T and rs538300761-T of LDLR; rs934197-T and rs9282606-A of APOB; and g.55038486A>G of PCSK9. In vitro assays revealed that rs36218923-T variant caused a significant mean reduction in luciferase activity (75.4% ± 2.9% SEM) compared to the construct containing the wild-type LDLR promoter, whereas the rs934197-T variant did not cause a significant difference in mean luciferase activity compared to the construct containing the wild-type APOB promoter. These results suggest that the rs36218923-T variant may be contributing to the decrease in LDLR gene expression and causing the FH phenotype. More studies corroborating these findings are needed to confirm the biological relevance of this variant.