Exploring the Genetic Predisposition to Epigenetic Changes in Alzheimer's Disease.

Alzheimer's disease (AD) is a prevalent type of dementia in elderly populations with a significant genetic component. The accumulating evidence suggests that AD involves a reconfiguration of the epigenetic landscape, including DNA methylation, post-translational modification of histone proteins, and chromatin remodeling. Along with environmental factors, individual specific genetic features play a considerable role in the formation of epigenetic architecture. In this study, we attempt to identify the non-coding regulatory SNPs (rSNPs) able to affect the epigenetic mechanisms in AD. To this end, the multi-omics approach is used. The GEO (Gene Expression Omnibus) available data (GSE153875) for AD patients and controls are integrated to reveal the rSNPs that display allele-specific features in both ChIP-seq profiles of four histone modifications and RNA-seq. Furthermore, we analyze the presence of rSNPs in the promoters of genes reported to be differentially expressed between AD and the normal brain (AD-related genes) and involved in epigenetic regulation according to the EpiFactors database. We also searched for the rSNPs in the promoters of the genes coding for transcription regulators of the identified AD-related genes. These regulators were selected based on the corresponding ChIP-seq peaks (ENCODE) in the promoter regions of these genes. Finally, we formed a panel of rSNPs localized to the promoters of genes that contribute to the epigenetic landscape in AD and, thus, to the genetic predisposition for this disease.

A Panel of rSNPs Demonstrating Allelic Asymmetry in Both ChIP-seq and RNA-seq Data and the Search for Their Phenotypic Outcomes through Analysis of DEGs.

Currently, the detection of the allele asymmetry of gene expression from RNA-seq data or the transcription factor binding from ChIP-seq data is one of the approaches used to identify the functional genetic variants that can affect gene expression (regulatory SNPs or rSNPs). In this study, we searched for rSNPs using the data for human pulmonary arterial endothelial cells (PAECs) available from the Sequence Read Archive (SRA). Allele-asymmetric binding and expression events are analyzed in paired ChIP-seq data for H3K4me3 mark and RNA-seq data obtained for 19 individuals. Two statistical approaches, weighted z-scores and predicted probabilities, were used to improve the efficiency of finding rSNPs. In total, we identified 14,266 rSNPs associated with both allele-specific binding and expression. Among them, 645 rSNPs were associated with GWAS phenotypes; 4746 rSNPs were reported as eQTLs by GTEx, and 11,536 rSNPs were located in 374 candidate transcription factor binding motifs. Additionally, we searched for the rSNPs associated with gene expression using an SRA RNA-seq dataset for 281 clinically annotated human postmortem brain samples and detected eQTLs for 2505 rSNPs. Based on these results, we conducted Gene Ontology (GO), Disease Ontology (DO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses and constructed the protein-protein interaction networks to represent the top-ranked biological processes with a possible contribution to the phenotypic outcome.

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

BACKGROUND: A 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. RESULTS: As 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. CONCLUSIONS: Overall, 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.

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.

BACKGROUND: Germline 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. RESULTS: First, 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 activity CONCLUSION: Our 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.

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 greater than A, rs12226937: G greater than A, and rs61761074: T greater than 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 greater than 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 greater than G substitution at nucleotide position 615 in the second intron of the KRAS gene (rs61761074) may represent a promising genetic marker of NSCLC.

Application of quantitative trait locus mapping and transcriptomics to studies of the senescence-accelerated phenotype in rats.

BACKGROUND: Etiology of complex disorders, such as cataract and neurodegenerative diseases including age-related macular degeneration (AMD), remains poorly understood due to the paucity of animal models, fully replicating the human disease. Previously, two quantitative trait loci (QTLs) associated with early cataract, AMD-like retinopathy, and some behavioral aberrations in senescence-accelerated OXYS rats were uncovered on chromosome 1 in a cross between OXYS and WAG rats. To confirm the findings, we generated interval-specific congenic strains, WAG/OXYS-1.1 and WAG/OXYS-1.2, carrying OXYS-derived loci of chromosome 1 in the WAG strain. Both congenic strains displayed early cataract and retinopathy but differed clinically from OXYS rats. Here we applied a high-throughput RNA sequencing (RNA-Seq) strategy to facilitate nomination of the candidate genes and functional pathways that may be responsible for these differences and can contribute to the development of the senescence-accelerated phenotype of OXYS rats. RESULTS: First, the size and map position of QTL-derived congenic segments were determined by comparative analysis of coding single-nucleotide polymorphisms (SNPs), which were identified for OXYS, WAG, and congenic retinal RNAs after sequencing. The transferred locus was not what we expected in WAG/OXYS-1.1 rats. In rat retina, 15442 genes were expressed. Coherent sets of differentially expressed genes were identified when we compared RNA-Seq retinal profiles of 20-day-old WAG/OXYS-1.1, WAG/OXYS-1.2, and OXYS rats. The genes most different in the average expression level between the congenic strains included those generally associated with the Wnt, integrin, and TGF-ß signaling pathways, widely involved in neurodegenerative processes. Several candidate genes (including Arhgap33, Cebpg, Gtf3c1, Snurf, Tnfaip3, Yme1l1, Cbs, Car9 and Fn1) were found to be either polymorphic in the congenic loci or differentially expressed between the strains. These genes may contribute to the development of cataract and retinopathy. CONCLUSIONS: This study is the first RNA-Seq analysis of the rat retinal transcriptome generated with 40 mln sequencing read depth. The integration of QTL and transcriptomic analyses in our study forms the basis of future research into the relationship between the candidate genes within the congenic regions and specific changes in the retinal transcriptome as possible causal mechanisms that underlie age-associated disorders.

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.

Potentialities of aberrantly methylated circulating DNA for diagnostics and post-treatment follow-up of lung cancer patients.

To date, aberrant DNA methylation has been shown to be one of the most common and early causes of malignant cell transformation and tumors of different localizations, including lung cancer. Cancer cell-specific methylated DNA has been found in the blood of cancer patients, indicating that cell-free DNA circulating in the blood (cirDNA) is a convenient tumor-associated DNA marker that can be used as a minimally invasive diagnostic test. In the current study, we investigated the methylation status in blood samples of 32 healthy donors and 60 lung cancer patients before and after treatment with neoadjuvant chemotherapy followed by total tumor resection. Using quantitative methylation-specific PCR, we found that the index of methylation (IM), calculated as IM = 100 × [copy number of methylated/(copy number of methylated + unmethylated gene)], for the RASSF1A and RARB2 genes in the cirDNA isolated from blood plasma and cell-surface-bound cirDNA was elevated 2- to 3-fold in lung cancer patients compared with healthy donors. Random forest classification tree model based on these variables combined (RARB2 and RASSF1A IM in both plasma and cell-surface-bound cirDNA) lead to NSCLC patients' and healthy subjects' differentiation with 87% sensitivity and 75% specificity. An association of increased IM values with an advanced stage of non-small-cell lung cancer was found for RARB2 but not for RASSF1A. Chemotherapy and total tumor resection resulted in a significant decrease in the IM for RARB2 and RASSF1A, in both cirDNA fractions, comparable to the IM level of healthy subjects. Importantly, a rise in the IM for RARB2 was detected in patients within the follow-up period, which manifested in disease relapse at 9 months, confirmed with instrumental and pathologic methods. Our data indicate that quantitative analysis of the methylation status of the RARB2 and RASSF1A tumor suppressor genes in both cirDNA fractions is a useful tool for lung cancer diagnostics, evaluation of cancer treatment efficiency and post-treatment monitoring.

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 0.32-0.47 g/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.