L. O. Bryzgalov

Detection of regulatory SNPs in human genome using ChIP-seq ENCODE data.

A vast amount of SNPs derived from genome-wide association studies are represented by non-coding ones, therefore exacerbating the need for effective identification of regulatory SNPs (rSNPs) among them. However, this task remains challenging since the regulatory part of the human genome is annotated much poorly as opposed to coding regions. Here we describe an approach aggregating the whole set of ENCODE ChIP-seq data in order to search for rSNPs, and provide the experimental evidence of its efficiency. Its algorithm is based on the assumption that the enrichment of a genomic region with transcription factor binding loci (ChIP-seq peaks) indicates its regulatory function, and thereby SNPs located in this region are more likely to influence transcription regulation. To ensure that the approach preferably selects functionally meaningful SNPs, we performed enrichment analysis of several human SNP datasets associated with phenotypic manifestations. It was shown that all samples are significantly enriched with SNPs falling into the regions of multiple ChIP-seq peaks as compared with the randomly selected SNPs. For experimental verification, 40 SNPs falling into overlapping regions of at least 7 TF binding loci were selected from OMIM. The effect of SNPs on the binding of the DNA fragments containing them to the nuclear proteins from four human cell lines (HepG2, HeLaS3, HCT-116, and K562) has been tested by EMSA. A radical change in the binding pattern has been observed for 29 SNPs, besides, 6 more SNPs also demonstrated less pronounced changes. Taken together, the results demonstrate the effective way to search for potential rSNPs with the aid of ChIP-seq data provided by ENCODE project.

Species‐specific effects of the hepatocarcinogens 3′‐methyl‐4‐dimethyl‐aminoazobenzene and ortho‐aminoazotoluene in mouse and rat liver

The effects of rat‐specific hepatocarcinogen 3′‐methyl‐4‐dimethylaminoazobenzene (3′‐MeDAB), mouse‐specific hepatocarcinogen ortho‐aminoazotoluene (OAT), non‐species‐specific hepatocarcinogen diethylnitrosamine (DENA), and non‐carcinogenic 4′‐methyl‐4‐dimethylaminoazobenzene (4′‐MeDAB) on glucocorticoid induction of tyrosine aminotransferase (TAT) and DNA‐binding activity of hepatocyte nuclear factor 3 (HNF3) family of transcription factors were investigated with carcinogen‐susceptible and ‐resistant animals. Species‐specific hepatocarcinogens 3′‐MeDAB and OAT strongly inhibited glucocorticoid induction of TAT in the liver of susceptible but not resistant animals. DENA, which is highly carcinogenic for the liver of both rats and mice inhibited glucocorticoid induction of TAT in both species, while non‐carcinogenic 4′‐MeDAB was absolutely ineffective both in rats and mice. The inhibition of TAT activity by the carcinogens was due to reduced levels of TAT mRNA, which is most likely to be a result of the reduced rate of transcription initiation of the TAT gene. In all cases, the TAT inhibition was accompanied by significant reduction of DNA‐binding activity of the HNF3 transcription factor, which is known to be critical to glucocorticoid regulation of TAT gene. We also demonstrated that the described species‐specific effects of OAT and of 3′‐MeDAB on HNF3 DNA‐binding activity may be initiated not only by administration in vivo, but also by their direct administration to homogenate, intact nuclei or nuclear lysate, but not to nuclear extract fraction, obtained by precipitation with 0.32 g/mL of ammonium sulfate (Fraction I). We showed, that a factor responsible for this effect might be precipitated in 0.32–0.47g/mL interval of ammonium sulfate concentration. In contrast, non‐specific hepatocarcinogen DENA was effective upon being added directly to Fraction I, implying a different mechanism of its action.

Program complex SNP-MED for analysis of single-nucleotide polymorphism (SNP) effects on the function of genes associated with socially significant diseases

We describe development and application of the new SNP-MED modular software system, designed to examine the influence of single-nucleotide polymorphisms (SNPs) on the function of genes associated with the risk of socially significant diseases. The SNP-MED system includes Genomics, Proteomics, and Gene Networks’ software components, and the Information Resource Database.

rSNP_Guide-based evaluation of SNPs in promoters of the human APC and MLH1 genes associated with colon cancer

We examined six regulatory SNPs (Single Nucleotide Polymorphisms) of two human genes, APC and MLH1 (rs75996864, rs76241113, rs78037487, rs80112297, rs80313086, and rs1800734), using the previously developed rSNP_Guide technology to calculate the significance of the changes in the binding of the SNP region to a set of 40 transcription factors. For each SNP, all tested proteins were ranked according to the significance of the changes in their affinity to the allelic DNA sequences and evaluated using Student’s t-test. We found that rs75996864, rs76241113, rs78037487, rs80112297, and rs80313086 alleles of the APC, as well as rs1800734 of the MLH1, promote stronger binding of the transcription factors, NF-Y, NFkB, c-Myb, RAR, YY-1, and Sp-1, all of which have been previously implicated in the progression of colon cancer. Our results substantiate further experimental analysis of the association between colon cancer and the five examined APC alleles (rs75996864, rs76241113, rs78037487, rs80112297, and rs80313086) using the commonly accepted human genetics methods.

Search for Regulatory SNPs Associated with Colon Cancer in the APC and MLH1 Genes

Polymorphisms belonging to the regulatory regions of the APC and MLH1 genes were detected by invoking ChIP-Seq data obtained in the ENCODE project. The significance of these polymorphisms for gene regulation was confirmed by gel retardation of DNA probes by nuclear proteins. More than half of the polymorphisms in the overlapping region of more than eight ChIP-Seq peaks were found to be significant for regulation.

Novel functional variants at the GWAS-implicated loci might confer risk to major depressive disorder, bipolar affective disorder and schizophrenia

BackgroundA challenge of understanding the mechanisms underlying cognition including neurodevelopmental and neuropsychiatric disorders is mainly given by the potential severity of cognitive disorders for the quality of life and their prevalence. However, the field has been focused predominantly on protein coding variation until recently. Given the importance of tightly controlled gene expression for normal brain function, the goal of the study was to assess the functional variation including non-coding variation in human genome that is likely to play an important role in cognitive functions. To this end, we organized and utilized available genome-wide datasets from genomic, transcriptomic and association studies into a comprehensive data corpus. We focused on genomic regions that are enriched in regulatory activity—overlapping transcriptional factor binding regions and repurpose our data collection especially for identification of the regulatory SNPs (rSNPs) that showed associations both with allele-specific binding and allele-specific expression. We matched these rSNPs to the nearby and distant targeted genes and then selected the variants that could implicate the etiology of cognitive disorders according to Genome-Wide Association Studies (GWAS). Next, we use DeSeq 2.0 package to test the differences in the expression of the certain targeted genes between the controls and the patients that were diagnosed bipolar affective disorder and schizophrenia. Finally, we assess the potential biological role for identified drivers of cognition using DAVID and GeneMANIA.ResultsAs a result, we selected fourteen regulatory SNPs locating within the loci, implicated from GWAS for cognitive disorders with six of the variants unreported previously. Grouping of the targeted genes according to biological functions revealed the involvement of processes such as ‘posttranscriptional regulation of gene expression’, ‘neuron differentiation’, ‘neuron projection development’, ‘regulation of cell cycle process’ and ‘protein catabolic processes’. We identified four rSNP-targeted genes that showed differential expression between patient and control groups depending on brain region: NRAS—in schizophrenia cohort, CDC25B, DDX21 and NUCKS1—in bipolar disorder cohort.ConclusionsOverall, our findings are likely to provide the keys for unraveling the mechanisms that underlie cognitive functions including major depressive disorder, bipolar disorder and schizophrenia etiopathogenesis.

Regulatory single nucleotide polymorphisms at the beginning of intron 2 of the human KRAS gene

There are two regulatory single nucleotide polymorphisms (rSNPs) at the beginning of the second intron of the mouse K-ras gene that are strongly associated with lung cancer susceptibility. We performed functional analysis of three SNPs (rs12228277: T>A, rs12226937: G>A, and rs61761074: T>G) located in the same region of human KRAS. We found that rs12228277 and rs61761074 result in differential binding patterns of lung nuclear proteins to oligonucleotide probes corresponding two alternative alleles; in both cases, the transcription factor NF-Y is involved. G>A substitution (rs12226937) had no effect on the binding of lung nuclear proteins. However, all the nucleotide substitutions under study showed functional effects in a luciferase reporter assay. Among them, rs61761074 demonstrated a significant correlation with allele frequency in non-small-cell lung cancer (NSCLC). Taken together, the results of our study suggest that a T>G substitution at nucleotide position 615 in the second intron of the KRAS gene (rs61761074) may represent a promising genetic marker of NSCLC.

Novel approach to functional SNPs discovery from genome-wide data reveals promising variants for colon cancer risk.

In the majority of colorectal cancer (CRC) cases, the genetic basis of predisposition remains unexplained. The goal of the study was to assess the regulatory SNPs (rSNPs) in the human genome and to reveal СRC drivers based on the available chromatin immunoprecipitation sequencing (ChIP‐Seq, ChIA‐PET) and transcriptional profiling (RNA‐Seq) data. We combined positional (locations within genome regulatory elements) and functional (associated with allele‐specific binding and expression) criteria followed by an analysis using genome‐wide association studies (GWAS) and minor allele frequency (MAF) datasets. DeSeq2 analysis through 70 CRC patients reinforced the regulatory potential. rSNPs (1,476) that were associated with significant (P < 0.01) allele‐specific events resulting in thirty that exhibited a link with CRC according to the MAF and 27, with a risk of malignancy in general according to GWAS. Selected rSNPs may modify the expression of genes for tumor suppressors and the regulators of signaling pathways, including noncoding RNAs. However, the rSNPs from the most represented group affect the expression of genes related to splicing. Our findings strongly suggest that the identified variants might contribute to CRC susceptibility, which indicates that aberrant splicing is one of the key mechanisms for unraveling disease etiopathogenesis and provides useful inputs for interpreting how genotypic variation corresponds to phenotypic outcome.

Regulatory single nucleotide polymorphisms (rSNPs) at the promoters 1A and 1B of the human APC gene

BackgroundGermline mutations in the coding sequence of the tumour suppressor APC gene give rise to familial adenomatous polyposis (which leads to colorectal cancer) and are associated with many other oncopathologies. The loss of APC function because of deletion of putative promoter 1A or 1B also results in the development of colorectal cancer. Since the regions of promoters 1A and 1B contain many single nucleotide polymorphisms (SNPs), the aim of this study was to perform functional analysis of some of these SNPs by means of an electrophoretic mobility shift assay (EMSA) and a luciferase reporter assay.ResultsFirst, it was shown that both putative promoters of APC (1A and 1B) drive transcription in an in vitro reporter experiment. From eleven randomly selected SNPs of promoter 1A and four SNPs of promoter 1B, nine and two respectively showed differential patterns of binding of nuclear proteins to oligonucleotide probes corresponding to alternative alleles. The luciferase reporter assay showed that among the six SNPs tested, the rs75612255 C allele and rs113017087 C allele in promoter 1A as well as the rs138386816 T allele and rs115658307 T allele in promoter 1B significantly increased luciferase activity in the human erythromyeloblastoid leukaemia cell line K562. In human colorectal cancer HCT-116 cells, none of the substitutions under study had any effect, with the exception of minor allele G of rs79896135 in promoter 1B. This allele significantly decreased the luciferase reporter’s activityConclusionOur results indicate that many SNPs in APC promoters 1A and 1B are functionally relevant and that allele G of rs79896135 may be associated with the predisposition to colorectal cancer.

Epigenetic probes for lung cancer monitoring: Line-1 methylation pattern in blood-circulating DNA

The main components of DNA aberrant methylation in malignant cells are promoter hypermethylation of specific genes and hypomethylation of the main part of DNA, in particular, the repeated sequences of retrotransposons. The hypomethylation of retrotransposons from the LINE-1 family was revealed in the lung cancer (LC) tissue. The composition of circulating DNA (cirDNA) from the plasma and blood cell surface bound circulating DNA (csb-cirDNA) was shown earlier to be altered in the blood of cancer patients due to the accumulation of tumor-specific aberrantly methylated DNA fragments, which are currently considered to be valuable cancer markers. The present study compares LINE-1 retrotransposon methylation patterns in the cirDNA and csb-cirDNA plasma from 21 untreated LC patients and 23 healthy donors. Concentrations of methylated LINE-1 region 1 copies (LINE-1met) were assessed by quantitative real-time methylation-specific PCR. In order to normalize the LINE-1 methylation level, the LINE-1 region 2 concentration was evaluated, which was independent of the methylation status (LINE-1Ind). The LINE1met concentration in csb-cirDNA tended to decrease (by a factor of 1.4) in blood from LC patients compared to healthy donors (Mann–Whitney test, P = 0.16). The LINE-1Ind (methylation-independent) concentration in csb-cirDNA was found to be lower by a factor of three in LC patients and by a factor of four in patients with adenocarcinoma than in healthy donors. Thus, along with the expected decrease in the LINE1met concentration in csb-cirDNA from LC patients’ blood, we recorded an unexpected statistically significant increase of the LINE-1 methylation index determined as (LINE-1met/LINE-1Ind) due to the profound decrease of the total LINE-1Ind number. There were no differences between the plasma cirDNA LINE-1 methylation indexes in the LC patients and healthy donors (Mann–Whitney test, P = 0.40). The data obtained agreed with our earlier results, which showed that csb-cirDNA was highly informative material for lung cancer diagnostics.