[Association of Polymorphic Genome Variants in the 2q32.1 Locus with the Development of Vasovagal Syncope].

The vasovagal syncope (VVS) is the most common form of syncope. The mechanisms of VVS development are not entirely clear. It is known that there is a genetic predisposition to this disease, but the data on the roles of individual genes are quite contradictory. Recently, a genome-wide association study identified a locus at chromosome 2q32.1 associated with a united group of diseases, that is, syncope and collapse; among the single nucleotide polymorphisms (SNPs) of this locus, the most significant association was observed for rs12465214. In a homogeneous sample of patients diagnosed with VVS, we analyzed the association of rs12465214, rs12621296, rs17582219 and rs1344706 located on chromosome 2q32.1 with this form of syncope. In the enrolled set, only rs12621296 was associated with VVS by itself, whereas associations of other SNPs were observed only in biallelic combinations. An epistatic interaction between the components of the combination rs12621296*A + rs17582219*A was revealed. The possible involvement of individual genes on the 2q32.1 locus in the genetic architecture of the VVS is discussed.

Multiple Sclerosis Is Associated with Immunoglobulin Germline Gene Variation of Transitional B Cells.

The regulatory functions of the B-cell compartment play an important role in the development and suppression of the immune response. Disruption of their anti-inflammatory functions may lead to the acceleration of immunopathological processes, and to autoimmune diseases, in particular. Unfortunately, the exact mechanism underlying the functioning and development of regulatory B cells (Breg) has not yet been fully elucidated. Almost nothing is known about their specificity and the structure of their B-cell receptors (BCRs). In this research, we analyzed the BCR repertoire of the transitional Breg (tBreg) subpopulation with the CD19+CD24highCD38high phenotype in patients with multiple sclerosis (MS), using next-generation sequencing (NGS). We show, for the first time, that the immunoglobulin germline distribution in the tBreg subpopulation is different between MS patients and healthy donors. The registered variation was more significant in patients with a more severe form of the disease, highly active MS (HAMS), compared to those with benign MS (BMS). Our data suggest that during MS development, deviations in the immunoglobulin Breg repertoire occur already at the early stage of B-cell maturation, namely at the stage of tBregs: between immature B cells in the bone marrow and mature peripheral B cells.

Senescence and entrenchment in evolution of amino acid sites.

Amino acid propensities at a site change in the course of protein evolution. This may happen for two reasons. Changes may be triggered by substitutions at epistatically interacting sites elsewhere in the genome. Alternatively, they may arise due to environmental changes that are external to the genome. Here, we design a framework for distinguishing between these alternatives. Using analytical modelling and simulations, we show that they cause opposite dynamics of the fitness of the allele currently occupying the site: it tends to increase with the time since its origin due to epistasis ("entrenchment"), but to decrease due to random environmental fluctuations ("senescence"). By analysing the genomes of vertebrates and insects, we show that the amino acids originating at negatively selected sites experience strong entrenchment. By contrast, the amino acids originating at positively selected sites experience senescence. We propose that senescence of the current allele is a cause of adaptive evolution.

Multilocus Analysis of Genetic Susceptibility to Myocardial Infarction in Russians: Replication Study.

In search of genetic markers of myocardial infarction (MI) risk, which have prognostic significance for Russians, we performed a replication study of MI association with genetic variants of PCSK9 (rs562556), APOE (epsilon polymorphism, rs7412 and rs429358), LPL (rs320), MTHFR (rs1801133), eNOS (rs2070744), and the 9p21 region (rs1333049) in 405 patients with MI and 198 controls. Significant MI association was observed with variants of the lipid metabolism genes (PCSK9, APOE and LPL), and of eNOS. The SNPs in the MTHFR gene and the 9p21 region were not significantly associated with MI one by one but were included in several different MI-associated allelic combinations identified by multilocus analysis. Since we have not revealed nonlinear epistatic interactions between the components of the identified combinations, we postulate that the cumulative effect of genes that form a combination arises from the summation of their small independent contributions. The prognostic significance of the additive composite model built from the PCSK9, APOE, LPL, and eNOS genes as genetic markers was assessed using ROC analysis. After we included these markers in the previously published composite model of individual genetic risk of MI, the prognostic efficacy in our sample reached AUC = 0.676. However, the results obtained in this study certainly need to be replicated in an independent sample of Russians.

Whole-Genome DNA Methylation Analysis of Peripheral Blood Mononuclear Cells in Multiple Sclerosis Patients with Different Disease Courses.

Multiple sclerosis (MS) is a severe neurodegenerative disease of polygenic etiology affecting the central nervous system. In addition to genetic factors, epigenetic mechanisms, primarily DNA methylation, which regulate gene expression, play an important role in MS development and progression. In this study, we have performed the first whole-genome DNA methylation profiling of peripheral blood mononuclear cells in relapsing-remitting MS (RRMS) and primary-progressive MS (PPMS) patients and compared them to those of healthy individuals in order to identify the differentially methylated CpG-sites (DMSs) associated with these common clinical disease courses. In addition, we have performed a pairwise comparison of DNA methylation profiles in RRMS and PPMS patients. All three pairwise comparisons showed significant differences in methylation profiles. Hierarchical clustering of the identified DMS methylation levels and principal component analysis for data visualization demonstrated a clearly defined aggregation of DNA samples of the compared groups into separate clusters. Compared with the control, more DMSs were identified in PPMS patients than in RRMS patients (67 and 30, respectively). More than half of DMSs are located in genes, exceeding the expected number for random distribution of DMSs between probes. RRMS patients mostly have hypomethylated DMSs, while in PPMS patients DMSs are mostly hypermethylated. CpG-islands and CpG-shores contain 60% of DMSs, identified by pairwise comparison of RRMS and control groups, and 79% of those identified by pairwise comparison of PPMS and control groups. Pairwise comparison of patients with two clinical MS courses revealed 51 DMSs, 82% of which are hypermethylated in PPMS. Overall, it was demonstrated that there are more changes in the DNA methylation profiles in PPMS than in RRMS. The data confirm the role of DNA methylation in MS development. We have shown, for the first time, that DNA methylation as an epigenetic mechanism is involved in the formation of two distinct clinical courses of MS: namely, RRMS and PPMS.

A review of genome-wide association studies for multiple sclerosis: classical and hypothesis-driven approaches.

Multiple sclerosis (MS) is a common complex neurodegenerative disease of the central nervous system. It develops with autoimmune inflammation and demyelination. Genome-wide association studies (GWASs) serve as a powerful tool for investigating the genetic architecture of MS and are generally used to identify the genetic factors of disease susceptibility, clinical phenotypes, and treatment response. This review considers the main achievements and challenges of using GWAS to identify the genes involved in MS. It also describes hypothesis-driven studies with extensive genome coverage of the selected regions, complementary to GWASs. To date, over 100 MS risk loci have been identified by the combination of both approaches; 40 of them were found in at least two GWASs and meet genome-wide significance threshold (p ≤ 5 × 10(-8)) in at least one GWAS, whereas the threshold for the rest of GWASs was set in our review at p < 1 × 10(-5). Yet, MS risk loci identified to date explain only a part of the total heritability, and the reasons of "missing heritability" are discussed. The functions of MS-associated genes are described briefly; the majority of them encode immune-response proteins involved in the main stages of MS pathogenesis.

GWAS-identified multiple sclerosis risk loci involved in immune response: validation in Russians.

Multiple sclerosis (MS) is a chronic neuro-inflammatory disease of complex etiology. The results of GWAS, a high-throughput method to discover genetic architecture of MS, require replication in independent ethnic groups. We performed a replication study of nine GWAS-identified SNPs in immune response in Russians. Associations of CLEC16A and IL2RA with MS were validated. Besides, we observed the associations of CLEC16A and IRF8 in women, and IL7RA and CD58 in men. With multi-locus association analysis two protective biallelic combinations: (TNFRSF1A*T+CLEC16A*A) and (TNFRSF1A*T+IRF8*A) were identified in women. Associations of CLEC16A*G/G and both biallelic combinations in women with MS survived the permutation test.

[The activation of the type I interferon signaling pathway in multiple sclerosis patients treated with russian analogue of Β-interferon-1b: transcriptome profiling data]. / Aktivatsiia signal'nogo puti interferonov tipa I u bol'nykh rasseiannym sklerozom pod vozdeistviem rossiiskogo analoga Β-interferona-1b (po dannym transkriptsionnogo profilirovaniia).

Implementation of the analogues of Β-interferon (Β-IFN), produced by recombinant strains of E. coli (Β-IFN-1b), for internal use in the Russian Federation for the treatment of multiple sclerosis (MS), implies a verification of their action at the transcriptome level in order to confirm the activation of the main signaling pathways involved in IFNb mechanism of action. In this work, the analysis is carried out for Infibeta (Generium, Russia). Using genome-wide transcriptional profiling with ILLUMINA HT-12 microarray, the differentially expressed genes in peripheral blood mononuclear cells of MS patients upon administration of Infibeta were studied. Comparison of gene expression levels in treatment-naive MS patients prior to first Β-IFN administration and 10 hours after it, identified 490 genes with significantly changed expression level, where 191 genes were up-regulated and 299 genes were down-regulated. Among the involved genes are those coding the components of the inflammatory system, innate and adaptive immunity, apoptosis, signal transduction, transcription, translation, degradation. Using gene set analysis, we confirmed the involvement of type I interferon signaling pathway genes and interferon-inducible genes in a patient's individual response to drug. Thus, the transcriptome profiling analysis allows concluding that the mechanism of action of Infibeta immunomodulatory drug is equal to that described for the original Β-IFN drugs.

[Genome-wide association study as a method for genetic architecture analysis in polygenic diseases (by the example of multiple sclerosis)].

Genome-wide association study (GWAS), which is a powerful tool for investigating the genetic architecture of polygenic disease in humans, is generally applied for identification of genetic factors of disease susceptibility, clinical phenotypes and treatment response. The differences in allele frequencies of genome-wide distributedsingle nucleotide polymorphisms (SNPs) are analyzed with microarray technique or other technologies, that allow simultaneous genotyping from tens'of thousands to several millions SNPs per sample. The power toexplore highly-reliable differences between compared groups of patients aind controls allowed GWAS to become a common approach for identification of genetic susceptibility for complex diseases with polygenic nature. The main achievements and challenges of GWAS in multiple sclerosis (MS), which is a prototype example of complex disease, are reviewed here for identification of MS causative genes, which expand the knowledge on molecular mechanisms of MS pathogenesis and genetic risk factors.

Polyreactive monoclonal autoantibodies in multiple sclerosis: functional selection from phage display library and characterization by deep sequencing analysis.

Multiple Sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system that primarily affects young and middle-aged people. It is widely accepted that B lymphocyte activation is required for MS progression. Despite the fact that the exact triggering mechanisms of MS remain enigmatic, one may suggest that MS can be induced by viral or bacterial infection in combination with specific genetic and environmental factors. Using deep sequencing and functional selection methodologies we characterized clones of poly- and cross-reactive antibodies that are capable of simultaneous recognition of viral proteins and autoantigens. The latter, in turn, possibly may trigger MS progression through molecular mimicry. It was identified that two cross-reactive antigens are probably recognized by light or heavy chains individually. According to the high structural homology between selected autoantibodies and a number of various antiviral IgGs, we suggest that a wide range of pathogens, instead of a single virus, be regarded as possible triggers of MS.

A Polygenic Approach to the Study of Polygenic Diseases.

Polygenic diseases are caused by the joint contribution of a number of independently acting or interacting polymorphic genes; the individual contribution of each gene may be small or even unnoticeable. The carriage of certain combinations of genes can determine the occurrence of clinically heterogeneous forms of the disease and treatment efficacy. This review describes the approaches used in a polygenic analysis of data in medical genomics, in particular, pharmacogenomics, aimed at identifying the cumulative effect of genes. This effect may result from the summation of gains of different genes or be caused by the epistatic interaction between the genes. Both cases are undoubtedly of great interest in investigating the nature of polygenic diseases. The means that allow one to discriminate between these two possibilities are discussed. The methods for searching for combinations of alleles of different genes associated with the polygenic phenotypic traits of the disease, as well as the methods for presenting and validating the results, are described and compared. An attempt is made to evaluate the applicability of the existing methods to an epistasis analysis. The results obtained by the authors using the APSampler software are described and summarized.

Contribution of the TGFB1 Gene to Myocardial Infarction Susceptibility.

Carriage frequencies of alleles and genotypes of theTGFB1 gene polymorphous loci -509C>T (rs1800469), 869T>C (rs1982073), 915G>C (rs1800471), which affect the level of cytokine TGF-ß1 production, were analyzed in the patients of Russian ethnic descent with myocardial infarction (MI) (406 cases) and in the control group of the same ethnic descent (198 controls). Significant association with MI was observed in carriage frequencies of the alleleTGFB1*-509T (p=0.046, OR =1.45, 95% CI: 1.02-2.06), genotypes TGFB1*869T/T (p=0.0024, OR =1.75, 95% CI: 1.22-2.51), andTGFB1*915G/G (p=0.048, OR=1.76, 95% CI: 1.05-2.97). Linkage disequilibrium analysis for these SNPs has shown that the associations revealed can be considered to be independent. A complex analysis of MI association with combinations of alleles/genotypes of said SNPs indicates their cumulative effect. An analysis of susceptibility to early-onset MI (≤ 50 years old) revealed a positive association of the alleleTGFB1*-509T (p=0.002, OR=2.24, 95% CI: 1.35-3.71) and genotypeTGFB1*869T/T (p=0.008, OR=1.93, 95% CI: 1.18-3.15), as well as their additivity. An analysis of susceptibility to recurrent MI revealed an association of the genotypeTGFB1*-509T/T (p=0.0078, OR=2.60, 95% CI: 1.28-5.28). The results obtained indicate the important role of theTGFB1gene in susceptibility to MI, including early-onset and recurrent MI, in Russians.

[Correlations between clusters of protein-DNA binding sites and the binding experimental data allow to predict a structure of regulatory modules].

Here we analyzed an option to predict the structure of cis-regulatory modules that consist of binding sites of different proteins (heterogeneous cis-regulatory modules) using mutual positional correlations between protein-DNA binding experimental data and computationally identified clusters of binding sites for each of the proteins.

[Polymorphic variants of genes encoding interleukin-6 and fibrinogen, the risk of ischemic stroke and fibrinogen levels].

Carriage frequencies of alleles and genotypes of polymorphous locus of -174G>C IL6 (rs1800795) were analyzed in the patients with ischemic stroke (IS) of Russian ethnic descent (200 cases) and in the control group of the same ethnic descent with similar sex and age (140 controls). Significant differences were identified in frequencies of carriage (in homo- or heterozygous form) of allele IL6*-174G (p = 0.0029, OR = 2.9, 95% CI: 1.4-5.8), which can be considered as risk factor for IS and in frequencies of IL6*-174C/C genotype carriage, correspondingly (p = 0.0029, OR = 0.35, 95% CI: 0.17-0.69). After sex stratification of patients and controls similar significant differences were observed only between female patients and controls, after age stratification the difference was observed only for the age group older 60 years. Complex analysis of association of SNP -174G>C IL6 alleles and genotypes carriage in combination with SNP 4266A>G (Thr312Ala) FGA (rs6050) (see symbol) -249C>T FGB (rs1800788) with IS revealed protective combinations IL6*-174C/C + FGA* 4266A (see symbol) IL6*-174C/C + FGB*-249C, which were slightly more significant than single protective genotype IL6*-174C/C associated with IS and their ORs didn't differ substantially from the single genotypes's OR value. At the same time the combinations of alternative allele IL6*-174G with the same FGB*-249C or FGA* 4266A alleles were revealed and their association significance levels as well as OR values were lower than the values for the single risk allele IL6*-174G. In case of the mutual carriage of IL6*-174G allele with FGA*4266A/A, FGB*-249C/C genotypes or with combinations of these alleles/genotypes the "neutralized" effect became stronger. In other words, we observed association of IS with allele/genotype combinations of genes IL6, FGA and FGB, in which IL6 plays key role and FGA and FGB have modulating function. In analysis of association of fibrinogen plasma levels with three analyzed polymorphous loci significant differences were not revealed.

[Splice sites are overrepresented in Pasilla binding motif clusters in D. melanogaster genes].

For RNA-binding protein Pasilla, which has been shown to play a role in alternative splicing regulation, binding sites and clusters of binding sites are found in silico in the whole genome of D. melanogaster. The current study analyzes the occurrence of splice sites in binding site clusters. Several hundred thousand binding site motifs and thousands of significant motif clusters were identified. It was discovered that exon-intron borders in D. melanogaster genes are reliably found within Pasilla binding motif clusters, with a higher frequency than could be otherwise expected based on a random model. Additionally, donor splice sites are found in Pasilla clusters twice as often as acceptor sites. This phenomena is observed both for exons annotated as alternatively spliced and for exons annotated as constitutive. These observations support the hypothesis that Pasilla plays a functional role in splicing regulation of D. melanogaster.

[Pharmacogenomics of multiple sclerosis: association of immune response genes polymorphism with copaxone treatment efficacy].

Complex association analysis of copaxone (glatiramer acetate) immunotherapy efficacy with allelic polymorphism in the number of immune response genes, which encode interferone beta (IFNB1), transforming growth factor beta1 (TGFB1), interferone gamma (IFNG), tumor necrosis factor (TNF), interferon alpha/beta receptor 1 (IFNAR1), CC chemokine receptor 5 (CCR5), interleukin 7 receptor alpha subunit (IL7RA), cytotoxic T-lymphocyte antigen 4 (CTLA4) and HLA class II histocompatibility antigen beta chain (DRB1) was performed with APSampler algorithm for 285 multiple sclerosis patients of Russian ethnicity. The results show evidence for the contribution of polymorphic variants in CCRS, DRB1, IFNG, TGFB1, IFNAR1, IL7RA and, probably, TNF and CTLA4 genes to copaxone treatment response. Single alleles of CCR5 and DRB1 genes are reliably associated with treatment efficacy. Carriage of allelic variants of other above mentioned genes contribute with reliable effect to copaxone treatment response as part of bi- and three-allelic combinations only. Present investigation may support basis toward the future possibility of prognostic test realization, which can provide a personal choice of immunomodulatory treatment for a patient with multiple sclerosis.

Deep and wide digging for binding motifs in ChIP-Seq data.

SUMMARY: ChIP-Seq data are a new challenge for motif discovery. Such a data typically consists of thousands of DNA segments with base-specific coverage values. We present a new version of our DNA motif discovery software ChIPMunk adapted for ChIP-Seq data. ChIPMunk is an iterative algorithm that combines greedy optimization with bootstrapping and uses coverage profiles as motif positional preferences. ChIPMunk does not require truncation of long DNA segments and it is practical for processing up to tens of thousands of data sequences. Comparison with traditional (MEME) or ChIP-Seq-oriented (HMS) motif discovery tools shows that ChIPMunk identifies the correct motifs with the same or better quality but works dramatically faster. AVAILABILITY AND IMPLEMENTATION: ChIPMunk is freely available within the ru_genetika Java package: http://line.imb.ac.ru/ChIPMunk. Web-based version is also available. CONTACT: ivan.kulakovskiy@gmail.com SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

[Complex analsis of association of inflammation genes with myocardial infarction].

Carriage frequencies of alleles and genotypes of functionally important polymorphous loci of some inflammation genes: proinflammatory cytokines genes IL-6, LTA and TNF, anti-inflammatory cytokine gene TGFB1 and CC chemokine receptor 5 gene CCR5 were analyzed in the patients with myocardial infarction (MI) of Russian ethnic descent (199 cases) and in the control group of the same ethnic descent (142 controls). Complex analysis using APSampler algorithm revealed MI association with carriage of all polymorphous variants studied, as individual risk factors (insertion/deletion polymorphism of CCR5 and SNP G252A LTA) or only in combination with other alleles/genotypes. Carriage of bi- or triallelic combinations was associated with MI more significantly than carriage of any their subsets: single alleles or allele pairs. Protective triallelic combination d*CCR5 + 252G*LTA(+) -174C*Ll-6 was found to be most significant (p = 0.0006, OR = 0.23, CI = 0.090-0.56). Separate analysis of genetic susceptibility to MI for men and women displayed sexual dimorphism for CCR5 gene.

[Asymmetry of the GC content in vicinity of transcription starts (with participation of polymerase PoII) and its correlation with location of adsorption sites of protein SP1 on DNA].

Nucleotide DNA sequences within the clusters of transcription starts, determined by the method of cap analysis of gene expression, have some distinguishing features. The sequences of these clusters are rich in nucleotides C and G, and there is an asymmetry of the nucleotide content, which correlates with the choice of chain from which the transcription in the cluster occurs. On the coding chain, the concentration of guanine exceeds the concentration of cytosine. In the nucleotide sequence of the cluster on the coding chain, the frequency of the polynucleotide tracts of the avoided nucleotide (cytosine), normalized to the frequency expected based on the content of this nucleotide in the cluster, is significantly higher compared with the normalized frequency of the polynucleotide tracts of the other nucleotide (guanine). Similarly, the statistical significance of the C-rich variant of the site of specific binding of the transcription initiation factor Sp1 in the coding chain is higher than that of the G-rich variant. However, the assumption can hardly be confirmed that the choice of the variant of the binding region of protein Sp1 correlates with the choice of the transcription chain. It is more likely that both variants are more or less equally probable, and the binding region of protein Sp1 acts in this case as a factor of selection, which counteracts the mutations inducing the shift of the nucleotide content.

[Genetic predisposition to multiple sclerosis as a polygenic autoimmune disease].

Unrelated patients with definite multiple sclerosis (MS) and healthy controls of Russian descent were genotyped at 16 polymorphic loci of the DRB1, TNF, LTa, TGFb1, CCR5 and CTLA4 genes and TNFa and TNFb microsatellites. The association of allelic variants with MS (p<0,01) was studied using the case-control method with the PSampler algorithm recently developed by our group. The previously described DRB1*15(2) allele, the TNFa9 allele and the biallelic combination (CCR5d32,DRB1*04) were reidentified as MS-associated in Russians. We also identified previously unknown MS-associated tri-allelic combinations: (-509 TGFb1*C, DRB1*18(3),CTLA4*G) and (-238TNF*B1, -308TNF*A2,CTLA4*G). The biological properties of the MS-associated genes support the notion that autoimmune inflammatory processes play an important role in MS, whereas an existence of mainly non-overlapping subgroups of patients bearing different predisposing genetic factors is consistent with the MS genetic heterogeneity.