Elena V. Kashina

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.

Identification of microRNA Genes in Three Opisthorchiids

Background Opisthorchis felineus, O. viverrini, and Clonorchis sinensis (family Opisthorchiidae) are parasitic flatworms that pose a serious threat to humans in some countries and cause opisthorchiasis/clonorchiasis. Chronic disease may lead to a risk of carcinogenesis in the biliary ducts. MicroRNAs (miRNAs) are small noncoding RNAs that control gene expression at post-transcriptional level and are implicated in the regulation of various cellular processes during the parasite- host interplay. However, to date, the miRNAs of opisthorchiid flukes, in particular those essential for maintaining their complex biology and parasitic mode of existence, have not been satisfactorily described. Methodology/Principal Findings Using a SOLiD deep sequencing-bioinformatic approach, we identified 43 novel and 18 conserved miRNAs for O. felineus (miracidia, metacercariae and adult worms), 20 novel and 16 conserved miRNAs for O. viverrini (adult worms), and 33 novel and 18 conserved miRNAs for C. sinensis (adult worms). The analysis of the data revealed differences in the expression level of conserved miRNAs among the three species and among three the developmental stages of O. felineus. Analysis of miRNA genes revealed two gene clusters, one cluster-like region and one intronic miRNA in the genome. The presence and structure of the two gene clusters were validated using a PCR-based approach in the three flukes. Conclusions This study represents a comprehensive description of miRNAs in three members of the family Opistorchiidae, significantly expands our knowledge of miRNAs in multicellular parasites and provides a basis for understanding the structural and functional evolution of miRNAs in these metazoan parasites. Results of this study also provides novel resources for deeper understanding the complex parasite biology, for further research on the pathogenesis and molecular events of disease induced by the liver flukes. The present data may also facilitate the development of novel approaches for the prevention and treatment of opisthorchiasis/clonorchiasis.

Candidate SNP markers of aggressiveness-related complications and comorbidities of genetic diseases are predicted by a significant change in the affinity of TATA-binding protein for human gene promoters

BackgroundAggressiveness in humans is a hereditary behavioral trait that mobilizes all systems of the body—first of all, the nervous and endocrine systems, and then the respiratory, vascular, muscular, and others—e.g., for the defense of oneself, children, family, shelter, territory, and other possessions as well as personal interests. The level of aggressiveness of a person determines many other characteristics of quality of life and lifespan, acting as a stress factor. Aggressive behavior depends on many parameters such as age, gender, diseases and treatment, diet, and environmental conditions. Among them, genetic factors are believed to be the main parameters that are well-studied at the factual level, but in actuality, genome-wide studies of aggressive behavior appeared relatively recently. One of the biggest projects of the modern science—1000 Genomes—involves identification of single nucleotide polymorphisms (SNPs), i.e., differences of individual genomes from the reference genome. SNPs can be associated with hereditary diseases, their complications, comorbidities, and responses to stress or a drug. Clinical comparisons between cohorts of patients and healthy volunteers (as a control) allow for identifying SNPs whose allele frequencies significantly separate them from one another as markers of the above conditions. Computer-based preliminary analysis of millions of SNPs detected by the 1000 Genomes project can accelerate clinical search for SNP markers due to preliminary whole-genome search for the most meaningful candidate SNP markers and discarding of neutral and poorly substantiated SNPs.ResultsHere, we combine two computer-based search methods for SNPs (that alter gene expression) {i} Web service SNP_TATA_Comparator (DNA sequence analysis) and {ii} PubMed-based manual search for articles on aggressiveness using heuristic keywords. Near the known binding sites for TATA-binding protein (TBP) in human gene promoters, we found aggressiveness-related candidate SNP markers, including rs1143627 (associated with higher aggressiveness in patients undergoing cytokine immunotherapy), rs544850971 (higher aggressiveness in old women taking lipid-lowering medication), and rs10895068 (childhood aggressiveness-related obesity in adolescence with cardiovascular complications in adulthood).ConclusionsAfter validation of these candidate markers by clinical protocols, these SNPs may become useful for physicians (may help to improve treatment of patients) and for the general population (a lifestyle choice preventing aggressiveness-related complications).

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.

The morphofunctional and biochemical characteristics of opisthorchiasis-associated cholangiocarcinoma in a Syrian hamster model

The validity of the experimental models of pathologies is one of the key challenges in translational medicine. Cholangiocarcinoma, or bile duct cancer, ranks second among oncological diseases of the liver. There is a strong association between bile duct cancer and parasitic infestation of the liver caused by trematodes in the Opisthorchiidae family. We recently demonstrated that cholangiocarcinoma can develop in Syrian hamsters (Mesocricetus auratus) infected by Opisthorchis felineus and administered with dimethylnitrosamine. However, there is still no description of how this experimental model can be used in translational research. The aim of this work was to study the morphological, functional, and biochemical characteristics during cholangiocarcinoma development in Syrian hamsters infected by O. felineus and administered with dimethylnitrosamine. The experiment lasted 30 weeks with combined administration of dimethylnitrosamine in drinking water at a dose of 12.5 ppm and a single injection of 50 metacercariae O. felineus. It was shown that the development of cholangiocarcinoma (18 weeks) increased the total number of basophils, eosinophils, and monocytes; the relative number of granulocytes, as well as the amount of total and direct bilirubin; and the cholesterol and ALT levels; however, it reduced the relative number of lymphocytes. Based on pathological, morphometric, and biochemical analyses, our model has characteristics similar to those in patients with opisthorchiasis-associated cholangiocarcinoma. Thus, this model can be used to test anticancer drugs, to study the mechanisms of cholangiocarcinogenesis, and to search for molecular markers for early diagnosis of bile duct 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>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.

Dynamics of IL-12 cytokine expression in human macrophages after dioxin exposure

Interleukin IL-12 is a key proinflammatory cytokine synthesized by macrophages; however, information on the effect of dioxin on its expression is still fragmentary. The presence of previously identified potential dioxin-responsive elements (DREs) in the regulatory regions of IL12A and IL12B genes, encoding IL-12 subunits IL-12p35 and IL-12p40, respectively, suggests direct activation of these genes by binding of the dioxin/AhR/ARNT complex to DREs. In this work the binding capacity of these DREs was demonstrated by gel shift assay. The study of the dynamics of IL12A and IL12B gene expression in the human U937 macrophages revealed no influence of dioxin on IL12A expression. In contrast, activation of IL12B gene expression with subsequent inhibition was noted. The observed dynamics can be explained by direct activation of the expression by the dioxin-containing complex and subsequent inhibition of the expression as a result of oxidative stress caused by dioxin. Thus, the well-known effect of dioxin on the immune system can be associated with its differential influence on the expression dynamics of the genes encoding IL-12 subunits.

Experimental opisthorchiasis: Study of blood cell composition, hematopoiesis, and startle reflex in laboratory animals

The Opisthorchis felineus (O. felineus), one of the species from the Opisthorchiidae family, causes severe disorders in the human and animal organism; therefore, it is the subject of topical scientific studies. In this work, a comparative study of the influence of the O. felineus invasions on the change in the blood cell composition, bone marrow hematopoiesis, and behavioral startle reflex was conducted in the C57BL/6 mice and Syrian hamsters two weeks after infection. Significant interspecies differences in many indices were found. It was established that the relative weight of the spleen (the main peripheral immune system organ) is significantly larger in mice than in hamsters. Moreover, the O. felineus invasion caused the hypertrophy of the spleen only in mice. More pronounced deviations from the norm in the blood cell composition, that were accompanied by the activation of myelo- and erythropoiesis, were observed in hamsters. The established changes in the blood in mice were not accompanied by changes in the bone marrow colony-forming activity. Mice also differed from hamsters by the startle reaction to the acoustic signal both by the severity of the reaction and prepulse inhibition value. No habituation reaction to the sound signal was observed in the infected hamsters. In addition, maturation of the O. felineus worms in hamsters occurred earlier than in mice. The data obtained indicate the increased resistance of mice to the O. feluneus infection; however, they do not exclude the possibility of using mice as a model during the study of the processes occurring in the host organism against the background of the experimental opisthorchiasis development.

Candidate SNP Markers of Familial and Sporadic Alzheimer's Diseases Are Predicted by a Significant Change in the Affinity of TATA-Binding Protein for Human Gene Promoters

While year after year, conditions, quality, and duration of human lives have been improving due to the progress in science, technology, education, and medicine, only eight diseases have been increasing in prevalence and shortening human lives because of premature deaths according to the retrospective official review on the state of US health, 1990-2010. These diseases are kidney cancer, chronic kidney diseases, liver cancer, diabetes, drug addiction, poisoning cases, consequences of falls, and Alzheimers disease (AD) as one of the leading pathologies. There are familial AD of hereditary nature (~4% of cases) and sporadic AD of unclear etiology (remaining ~96% of cases; i.e., non-familial AD). Therefore, sporadic AD is no longer a purely medical problem, but rather a social challenge when someone asks oneself: “What can I do in my own adulthood to reduce the risk of sporadic AD at my old age to save the years of my lifespan from the destruction caused by it?” Here, we combine two computational approaches for regulatory SNPs: Web service SNP_TATA_Comparator for sequence analysis and a PubMed-based keyword search for articles on the biochemical markers of diseases. Our purpose was to try to find answers to the question: “What can be done in adulthood to reduce the risk of sporadic AD in old age to prevent the lifespan reduction caused by it?” As a result, we found 89 candidate SNP markers of familial and sporadic AD (e.g., rs562962093 is associated with sporadic AD in the elderly as a complication of stroke in adulthood, where natural marine diets can reduce risks of both diseases in case of the minor allele of this SNP). In addition, rs768454929, and rs761695685 correlate with sporadic AD as a comorbidity of short stature, where maximizing stature in childhood and adolescence as an integral indicator of health can minimize (or even eliminate) the risk of sporadic AD in the elderly. After validation by clinical protocols, these candidate SNP markers may become interesting to the general population [may help to choose a lifestyle (in childhood, adolescence, and adulthood) that can reduce the risks of sporadic AD, its comorbidities, and complications in the elderly].