The Search for and Functional Analysis of Genetic Variants in microRNA-Binding Sites using Massively Parallel Reporter Assay.

A large-scale search for the genetic variants with a bias in the representation of alleles in transcriptome data (AE SNPs) and the binding sites in microRNA 3'-UTRs was performed and their functional significance was assessed using massively parallel reporter assay (MPRA). Of the 629,559 associated "SNP-gene" pairs (eQTLs) discovered in the human liver tissue according to the GTEx Analysis V8 data, 4394 polymorphic positions in the 3'-UTRs of the genes, which represent the eQTLs for these genes were selected. The TargetScanHuman 7.0 algorithm and PolymiRTS database were searched for the potential microRNA-binding sites. Of the predicted microRNA sites affected by eQTL-SNPs, we selected 51 sites with the best evidence of functionality according to Ago2-CLIP-seq, CLEAR-CLIP, and eCLIP-seq for RNA-binding proteins. For MPRA, a library of the plasmids carrying the main and alternative alleles for each AE SNP (in total, 102 constructs) was created. Allele-specific expression for 6 SNPs was detected by transfection of the HepG2 cell line with the constructed plasmid library and sequencing of target DNA and RNA sequences using the Illumina (MiSeq) platform.

Human-genome single nucleotide polymorphisms affecting transcription factor binding and their role in pathogenesis.

Single nucleotide polymorphisms (SNPs) are the most common type of variation in the human genome. The vast majority of SNPs identified in the human genome do not have any effect on the phenotype; however, some can lead to changes in the function of a gene or the level of its expression. Most SNPs associated with certain traits or pathologies are mapped to regulatory regions of the genome and affect gene expression by changing transcription factor binding sites. In recent decades, substantial effort has been invested in searching for such regulatory SNPs (rSNPs) and understanding the mechanisms by which they lead to phenotypic differences, primarily to individual differences in susceptibility to diseases and in sensitivity to drugs. The development of the NGS (next-generation sequencing) technology has contributed not only to the identification of a huge number of SNPs and to the search for their association (genome-wide association studies, GWASs) with certain diseases or phenotypic manifestations, but also to the development of more productive approaches to their functional annotation. It should be noted that the presence of an association does not allow one to identify a functional, truly disease-associated DNA sequence variant among multiple marker SNPs that are detected due to linkage disequilibrium. Moreover, determination of associations of genetic variants with a disease does not provide information about the functionality of these variants, which is necessary to elucidate the molecular mechanisms of the development of pathology and to design effective methods for its treatment and prevention. In this regard, the functional analysis of SNPs annotated in the GWAS catalog, both at the genome-wide level and at the level of individual SNPs, became especially relevant in recent years. A genome-wide search for potential rSNPs is possible without any prior knowledge of their association with a trait. Thus, mapping expression quantitative trait loci (eQTLs) makes it possible to identify an SNP for which - among transcriptomes of homozygotes and heterozygotes for its various alleles - there are differences in the expression level of certain genes, which can be located at various distances from the SNP. To predict rSNPs, approaches based on searches for allele-specific events in RNA-seq, ChIP-seq, DNase-seq, ATAC-seq, MPRA, and other data are also used. Nonetheless, for a more complete functional annotation of such rSNPs, it is necessary to establish their association with a trait, in particular, with a predisposition to a certain pathology or sensitivity to drugs. Thus, approaches to finding SNPs important for the development of a trait can be categorized into two groups: (1) starting from data on an association of SNPs with a certain trait, (2) starting from the determination of allele-specific changes at the molecular level (in a transcriptome or regulome). Only comprehensive use of strategically different approaches can considerably enrich our knowledge about the role of genetic determinants in the molecular mechanisms of trait formation, including predisposition to multifactorial diseases.

rs2072580T>A Polymorphism in the Overlapping Promoter Regions of the SART3 and ISCU Genes Associated with the Risk of Breast Cancer.

We analyzed association of potentially regulatory polymorphisms (rs590352, rs11542583, rs3829202, rs207258, and rs4796672) with breast cancer. A significant association was found between this disease and rs2072580T>A (p=0.001) located in the overlapping promoter regions of the SART3 and ISCU genes. In women with AA and AT genotypes, the risk of breast cancer is higher by 6.7 times (p=0.001) and 12 times (p=0.001), respectively, in comparison with TT genotype. Under a codominant model of inheritance (AT vs AA+TT), the risk of breast cancer was increased by 4.2 times (р=0.001) for the AT genotype. Under a recessive model of inheritance (TT vs AA+TT), the risk of disease was 10-fold higher (р=0.001) for the TT genotype. It has been demonstrated that the T>A substitution affects the binding properties of transcription factors CREB1 and REST.