Circular RNAs: Biogenesis, Functions, and Role in Myocardial Hypertrophy.

Circular RNAs (circRNAs) are a large class of endogenous single-stranded covalently closed RNA molecules. High-throughput RNA sequencing and bioinformatic algorithms have identified thousands of eukaryotic circRNAs characterized by high stability and tissue-specific expression pattern. Recent studies have shown that circRNAs play an important role in the regulation of physiological processes in the norm and in various diseases, including cardiovascular disorders. The review presents current concepts of circRNA biogenesis, structural features, and biological functions, describes the methods of circRNA analysis, and summarizes the results of studies on the role of circRNAs in the pathogenesis of hypertrophic cardiomyopathy, the most common inherited heart disease.

Shared genetic architecture of COVID-19 and Alzheimer's disease.

The severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) and the сoronavirus disease 2019 (COVID-19) have become a global health threat. At the height of the pandemic, major efforts were focused on reducing COVID-19-associated morbidity and mortality. Now is the time to study the long-term effects of the pandemic, particularly cognitive impairment associated with long COVID. In recent years much attention has been paid to the possible relationship between COVID-19 and Alzheimer's disease, which is considered a main cause of age-related cognitive impairment. Genetic predisposition was shown for both COVID-19 and Alzheimer's disease. However, the analysis of the similarity of the genetic architecture of these diseases is usually limited to indicating a positive genetic correlation between them. In this review, we have described intrinsic linkages between COVID-19 and Alzheimer's disease, pointed out shared susceptibility genes that were previously identified in genome-wide association studies of both COVID-19 and Alzheimer's disease, and highlighted a panel of SNPs that includes candidate genetic risk markers of the long COVID-associated cognitive impairment.

Novel Genes Involved in Hypertrophic Cardiomyopathy: Data of Transcriptome and Methylome Profiling.

Hypertrophic cardiomyopathy (HCM) is the most common inherited heart disease; its pathogenesis is still being intensively studied to explain the reasons for the significant genetic and phenotypic heterogeneity of the disease. To search for new genes involved in HCM development, we analyzed gene expression profiles coupled with DNA methylation profiles in the hypertrophied myocardia of HCM patients. The transcriptome analysis identified significant differences in the levels of 193 genes, most of which were underexpressed in HCM. The methylome analysis revealed 1755 nominally significant differentially methylated positions (DMPs), mostly hypomethylated in HCM. Based on gene ontology enrichment analysis, the majority of biological processes, overrepresented by both differentially expressed genes (DEGs) and DMP-containing genes, are involved in the regulation of locomotion and muscle structure development. The intersection of 193 DEGs and 978 DMP-containing genes pinpointed eight common genes, the expressions of which correlated with the methylation levels of the neighboring DMPs. Half of these genes (AUTS2, BRSK2, PRRT1, and SLC17A7), regulated by the mechanism of DNA methylation, were underexpressed in HCM and were involved in neurogenesis and synapse functioning. Our data, suggesting the involvement of innervation-associated genes in HCM, provide additional insights into disease pathogenesis and expand the field of further research.

Male-specific coordinated changes in expression of miRNA genes, but not other genes within the DLK1-DIO3 locus in multiple sclerosis.

The role of miRNAs, small non-coding regulatory RNAs, in the molecular mechanisms of multiple sclerosis (MS) development has been intensively studied. MiRNAs tend to be clustered within imprinted regions, and the largest number of miRNA genes is observed in the DLK1-DIO3 locus. Earlier using RNA-seq we identified sex-specific upregulation of the set of miRNA genes from this locus in peripheral blood mononuclear cells (PBMC) of treatment-naive relapsing-remitting MS (RRMS) patients. In the present study we set up to independently investigate the expression of a vast array of genes present in the DLK1-DIO3 imprinted locus. First, we analyzed the expression of miRNA genes, which levels in RRMS were mostly inconsistent based on RNA-seq data and not previously explored using qPCR. We identified that all selected miRNAs - miR-337-3p and -665 from 14q32.2 cluster and miR-370c, -380, -494, -654-3p, -300, -539, -668, and -323b-5p - were upregulated in MS men, but not women when compared to controls, regardless of conflicting RNA-seq data. The expression of miRNAs from the DLK1-DIO3 locus was highly correlated, indicating the existence of a common regulatory mechanism(s) that controls miRNA expression, regardless of the position of their genes within this region. Second, we performed the expression analysis of non-miRNA genes within the locus. The genes encoding proteins (DLK1, DIO3, RTL1), long non-coding RNAs (MEG3, MEG8, and MEG9) and small nucleolar RNAs (SNORD112, SNORD113-5, SNORD113-7, SNORD114-3, SNORD114-8, SNORD114-19) were not dysregulated in RRMS both in men and women. DNA methylation analysis of selected CpG sites within the differentially methylated regions IG-DMR, MEG3-DMR, and MEG8-DMR of the DLK1-DIO3 imprinted locus pointed out that they were not involved in the regulation of miRNA gene expression in RRMS, at least in PBMC population. The question of whether the observed changes in expression of miRNA genes (given that there is a constant expression of other non-miRNA genes of the DLK1-DIO3 locus) are involved in the development of RRMS or are they a consequence of the disease progress, remains open and needs further investigation.

Circulating miR-499a-5p Is a Potential Biomarker of MYH7-Associated Hypertrophic Cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is the most common inherited myocardial disease with significant genetic and phenotypic heterogeneity. To search for novel biomarkers, which could increase the accuracy of HCM diagnosis and improve understanding of its phenotype formation, we analyzed the levels of circulating miRNAs­stable non-coding RNAs involved in post-transcriptional gene regulation. Performed high throughput sequencing of miRNAs in plasma of HCM patients and controls pinpointed miR-499a-5p as one of 35 miRNAs dysregulated in HCM. Further investigation on enlarged groups of individuals showed that its level was higher in carriers of pathogenic/likely pathogenic (P/LP) variants in MYH7 gene compared to controls (fold change, FC = 8.9; p < 0.0001). Just as important, carriers of variants in MYH7 gene were defined with higher miRNA levels than carriers of variants in the MYBPC3 gene (FC = 14.1; p = 0.0003) and other patients (FC = 4.1; p = 0.0008). The receiver operating characteristic analysis analysis showed the ability of miR-499a-5p to identify MYH7 variant carriers with the HCM phenotype with area under the curve value of 0.95 (95% confidence interval: 0.88−1.03, p = 0.0004); sensitivity and specificity were 0.86 and 0.91 (cut-off = 0.0014). Therefore, miR-499a-5p could serve as a circulating biomarker of HCM, caused by P/LP variants in MYH7 gene.

Genome-wide DNA methylation profiling identifies epigenetic changes in CD4+ and CD14+ cells of multiple sclerosis patients.

Multiple sclerosis (MS) is a chronic autoimmune and degenerative disease of the central nervous system, which develops in genetically predisposed individuals upon exposure to environmental influences. Environmental triggers of MS, such as viral infections or smoking, were demonstrated to affect DNA methylation, and thus to involve this important epigenetic mechanism in the development of pathological process. To identify MS-associated DNA methylation hallmarks, we performed genome-wide DNA methylation profiling of two cell populations (CD4+ T-lymphocytes and CD14+ monocytes), collected from the same treatment-naive relapsing-remitting MS patients and healthy subjects, using Illumina 450 K methylation arrays. We revealed significant changes in DNA methylation for both cell populations in MS. In CD4+ cells of MS patients the majority of differentially methylated positions (DMPs) were shown to be hypomethylated, while in CD14+ cells - hypermethylated. Differential methylation of HLA-DRB1 gene in CD4+ and CD14+ cells was associated with carriage of DRB1*15 allele independently from the disease status. Besides, about 20% of identified DMPs were shared between two cell populations and had the same direction of methylation changes; they may be involved in basic epigenetic processes occuring in MS. These findings suggest that the epigenetic mechanism of DNA methylation in immune cells contributes to MS; further studies are now required to validate these results and understand their functional significance.

Global transcriptome profiling in peripheral blood mononuclear cells identifies dysregulation of immune processes in individuals with radiologically isolated syndrome.

The presence of brain/spinal white matter lesions typical for multiple sclerosis (MS) in asymptomatic individuals is known as 'radiologically isolated syndrome' (RIS). Taking into account that RIS patients are at high risk of MS development, the understanding of mechanisms underlying its pathogenesis is of great importance. In order to investigate RIS-specific transcription signature we performed high-throughput RNA-sequencing in peripheral blood mononuclear cells (PBMCs) of 8 RIS patients and 8 age- and sex-matched healthy controls. We identified 57 differentially expressed genes (DEGs), which levels differed by more than 2 times when comparing RIS patients to healthy controls (FDR p value < 0.05). Gene ontology enrichment analysis in the "biological process" category revealed 16 signaling pathways significantly overrepresented by identified DEGs. The most significant changes in gene expression in PBMCs of RIS patients occur in pathways involved in regulation of the immune response, cytokine and chemokine signaling, cytokine production, and leukocyte migration. In general, analyzing the global transcriptome we demonstrated the dysregulation of immune processes in PBMCs of RIS patients, confirming the current assumption that RIS represents the preclinical stage and/or subclinical form of MS.

MiRNA-Regulated Pathways for Hypertrophic Cardiomyopathy: Network-Based Approach to Insight into Pathogenesis.

Hypertrophic cardiomyopathy (HCM) is the most common hereditary heart disease. The wide spread of high-throughput sequencing casts doubt on its monogenic nature, suggesting the presence of mechanisms of HCM development independent from mutations in sarcomeric genes. From this point of view, HCM may arise from the interactions of several HCM-associated genes, and from disturbance of regulation of their expression. We developed a bioinformatic workflow to study the involvement of signaling pathways in HCM development through analyzing data on human heart-specific gene expression, miRNA-target gene interactions, and protein-protein interactions, available in open databases. Genes regulated by a pool of miRNAs contributing to human cardiac hypertrophy, namely hsa-miR-1-3p, hsa-miR-19b-3p, hsa-miR-21-5p, hsa-miR-29a-3p, hsa-miR-93-5p, hsa-miR-133a-3p, hsa-miR-155-5p, hsa-miR-199a-3p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-451a, and hsa-miR-497-5p, were considered. As a result, we pinpointed a module of TGFß-mediated SMAD signaling pathways, enriched by targets of the selected miRNAs, that may contribute to the cardiac remodeling in HCM. We suggest that the developed network-based approach could be useful in providing a more accurate glimpse on pathological processes in the disease pathogenesis.

Imprinted Genes and Multiple Sclerosis: What Do We Know?

Multiple sclerosis (MS) is a chronic autoimmune neurodegenerative disease of the central nervous system that arises from interplay between non-genetic and genetic risk factors. The epigenetics functions as a link between these factors, affecting gene expression in response to external influence, and therefore should be extensively studied to improve the knowledge of MS molecular mechanisms. Among others, the epigenetic mechanisms underlie the establishment of parent-of-origin effects that appear as phenotypic differences depending on whether the allele was inherited from the mother or father. The most well described manifestation of parent-of-origin effects is genomic imprinting that causes monoallelic gene expression. It becomes more obvious that disturbances in imprinted genes at the least affecting their expression do occur in MS and may be involved in its pathogenesis. In this review we will focus on the potential role of imprinted genes in MS pathogenesis.

From miRNA Target Gene Network to miRNA Function: miR-375 Might Regulate Apoptosis and Actin Dynamics in the Heart Muscle via Rho-GTPases-Dependent Pathways.

MicroRNAs (miRNAs) are short, single-stranded, non-coding ribonucleic acid (RNA) molecules, which are involved in the regulation of main biological processes, such as apoptosis or cell proliferation and differentiation, through sequence-specific interaction with target mRNAs. In this study, we propose a workflow for predicting miRNAs function by analyzing the structure of the network of their target genes. This workflow was applied to study the functional role of miR-375 in the heart muscle (myocardium), since this miRNA was previously shown to be associated with heart diseases, and data on its function in the myocardium are mostly unclear. We identified PIK3CA, RHOA, MAPK3, PAFAH1B1, CTNNB1, MYC, PRKCA, ERBB2, and CDC42 as key genes in the miR-375 regulated network and predicted the possible function of miR-375 in the heart muscle, consisting mainly in the regulation of the Rho-GTPases-dependent signaling pathways. We implemented our algorithm for miRNA function prediction into a Python module, which is available at GitHub.

Mitonuclear interactions influence multiple sclerosis risk.

Multiple sclerosis (MS) is a chronic disease of the central nervous system characterized by the autoimmune inflammation, demyelination, and neurodegeneration. This complex disease develops in genetically predisposed individuals under adverse environmental factors. To date, a large number of MS-associated polymorphic loci of the nuclear genome have been identified; however, their total variability can explain only about 48% of the observed inheritance of MS. Polymorphic variants of the mitochondrial genome and interactions of mitochondrial and nuclear genes (mitonuclear interactions) may be the possible sources of the "missing heritability". We analyzed the association with MS of 10 mitochondrial DNA polymorphisms (m.1719, m.4216, m.4580, m.4917, m.7028, m.9055, m.10398, m.12308, m.13368, m.13708) in DNA of 540 MS patients and 406 healthy individuals. The allele m.9055*G was the only mitochondrial variant associated with MS (Pf = 0.027). To evaluate interactions of mitochondrial and nuclear genomes, we searched for biallelic combinations containing one of 10 mitochondrial variants and one of 35 variants of immune-related nuclear genes. Carriership of mitochondrial variants m.4216, m.4580, or m.13708 in biallelic combinations with variants of nuclear genes IL7R, CLEC16A, CD6, CD86 or PVT1 was associated with MS (Pf = 0.0036-0.00030). We identified epistatic interaction between components of a combination (m.13708*A + PVT1 rs4410871*T). The existence of epistatic biallelic combination can reflect the genuine mitonuclear epistasis.

Genetic differences between primary progressive and relapsing-remitting multiple sclerosis: The impact of immune-related genes variability.

BACKGROUND: Multiple sclerosis (MS) is a chronic autoimmune disease of CNS with a highly heterogeneous clinical course. The role of the genetic variability in determination of MS course is not yet well established. We aimed to estimate the impact of immune-related genes variability in the genetic architecture of two clinically different MS courses - primary progressive (PPMS) and relapsing-remitting (RRMS). METHODS: We performed the association analysis of 31 immune-related genes' variants in pairwise comparisons of 110 PPMS patients, 564 RRMS patients and 424 healthy individuals (HI). RESULTS: HLA-DRB1*11 and *15, IL7RA rs6897932*C/C, CXCR5 rs523604*A/A, and CLEC16A rs6498169*G/G were found as MS-associated variants common for PPMS and RRMS. HLA-DRB1*07, IL4 rs2243250*C/C, IRF5 rs4728142*A/A, and IFNAR2 rs2248202*C were PPMS- associated when compared to HI and RRMS, while HLA-DRB1*09 and IL6 rs1800795*C were RRMS-associated when compared to HI and PPMS. In multiple logistic regression and ROC curve analyses composite regression models were characterized by area under the curve values of 0.769, 0.726, and 0.679 in comparisons "PPMS vs HI", "RRMS vs HI", and "PPMS vs RRMS", respectively. CONCLUSION: We revealed genetic variants associated with PPMS, among which both variants common for two MS courses and distinguishing PPMS and RRMS were found. Observed genetic features of two MS courses may underlie different impact of autoimmune inflammatory processes in development of PPMS and RRMS, however additional studies on larger PPMS samples are strictly needed.

MiRNAs from DLK1-DIO3 Imprinted Locus at 14q32 are Associated with Multiple Sclerosis: Gender-Specific Expression and Regulation of Receptor Tyrosine Kinases Signaling.

Relapsing-remitting multiple sclerosis (RRMS) is the most prevalent course of multiple sclerosis. It is an autoimmune inflammatory disease of the central nervous system. To investigate the gender-specific involvement of microRNAs (miRNAs) in RRMS pathogenesis, we compared miRNA profiles in peripheral blood mononuclear cells separately in men and women (eight RRMS patients versus four healthy controls of each gender) using high-throughput sequencing. In contrast to women, six downregulated and 26 upregulated miRNAs (padj < 0.05) were identified in men with RRMS. Genes encoding upregulated miRNAs are co-localized in DLK1-DIO3 imprinted locus on human chromosome 14q32. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis was performed in independent groups of men (16 RRMS patients and 10 healthy controls) and women (20 RRMS patients and 10 healthy controls). Increased expression of miR-431, miR-127-3p, miR-379, miR-376c, miR-381, miR-410 and miR-656 was again demonstrated in male (padj < 0.05), but not in female RRMS patients. At the same time, the expression levels of these miRNAs were lower in healthy men than in healthy women, whereas in RRMS men they increased and reached or exceeded levels in RRMS women. In general, we demonstrated that expression levels of these miRNAs depend both on "health⁻disease" status and gender. Network-based enrichment analysis identified that receptor tyrosine kinases-activated pathways were enriched with products of genes targeted by miRNAs from DLK1-DIO3 locus. These results suggest the male-specific involvement of these miRNAs in RRMS pathogenesis via regulation of PI3K/Akt signaling.

Protective Allele for Multiple Sclerosis HLA-DRB1*01:01 Provides Kinetic Discrimination of Myelin and Exogenous Antigenic Peptides.

Risk of the development of multiple sclerosis (MS) is known to be increased in individuals bearing distinct class II human leukocyte antigen (HLA) variants, whereas some of them may have a protective effect. Here we analyzed distribution of a highly polymorphous HLA-DRB1 locus in more than one thousand relapsing-remitting MS patients and healthy individuals of Russian ethnicity. Carriage of HLA-DRB1*15 and HLA-DRB1*03 alleles was associated with MS risk, whereas carriage of HLA-DRB1*01 and HLA-DRB1*11 was found to be protective. Analysis of genotypes revealed the compensatory effect of risk and resistance alleles in trans. We have identified previously unknown MBP153-161 peptide located at the C-terminus of MBP protein and MBP90-98 peptide that bound to recombinant HLA-DRB1*01:01 protein with affinity comparable to that of classical antigenic peptide 306-318 from the hemagglutinin (HA) of the influenza virus demonstrating the ability of HLA-DRB1*01:01 to present newly identified MBP153-161 and MBP90-98 peptides. Measurements of kinetic parameters of MBP and HA peptides binding to HLA-DRB1*01:01 catalyzed by HLA-DM revealed a significantly lower rate of CLIP exchange for MBP153-161 and MBP90-98 peptides as opposed to HA peptide. Analysis of the binding of chimeric MBP-HA peptides demonstrated that the observed difference between MBP153-161, MBP90-98, and HA peptide epitopes is caused by the lack of anchor residues in the C-terminal part of the MBP peptides resulting in a moderate occupation of P6/7 and P9 pockets of HLA-DRB1*01:01 by MBP153-161 and MBP90-98 peptides in contrast to HA308-316 peptide. This leads to the P1 and P4 docking failure and rapid peptide dissociation and release of empty HLA-DM-HLA-DR complex. We would like to propose that protective properties of the HLA-DRB1*01 allele could be directly linked to the ability of HLA-DRB1*01:01 to kinetically discriminate between antigenic exogenous peptides and endogenous MBP derived peptides.

NGS-identified circulating miR-375 as a potential regulating component of myocardial infarction associated network.

Acute myocardial infarction (MI), the most severe type of coronary heart disease, is a leading cause of disability and mortality worldwide. In order to investigate the involvement of miRNAs in the pathologic processes related to MI, we performed the analysis of circulating miRNAs - stable short noncoding RNA molecules - in the peripheral blood plasma of MI patients compared to healthy controls (all persons were men and lived in European Russia) using next generation sequencing. We observed 20 miRNAs, which levels in plasma more than two-fold differed in MI patients (p < 0.05). Among them miR-208b and miR-375 passed threshold for multiple corrections (FC = 49.2, FDR-adjusted p-value = 0.0078 and FC = -6.4, FDR-adjusted p-value = 0.00076, respectively); these data were then validated using RT-qPCR (FC = 5.3, p-value = 0.028 and FC = -2.1, p-value = 0.0039, respectively). While for miR-208b we reidentified earlier observations, miR-375 was found to be associated with MI for the first time. To investigate the reasons for which miR-375 holds a special place among circulating miRNAs in MI, enrichment and network analyses of miR-375 target genes and their interactions were carried out. PIK3CA and TP53 genes, regulated by miR-375, were identified as the key players of MI disease module.

Immune-related miRNA expression patterns in peripheral blood mononuclear cells differ in multiple sclerosis relapse and remission.

MiRNAs were shown to participate in development of autoimmune inflammatory process in multiple sclerosis (MS). To investigate miRNAs involvement in relapse-remission MS course, we analyzed expression of immune-related miRNAs in PBMC of treatment-naïve relapsing and remitting MS patients and healthy controls. The upregulation of miR-126-3p, miR-146b-5p, miR-155, miR-196a-5p, miR-21-5p, miR-223-3p, miR-326 and miR-379-5p in remission compared to relapse was observed; when apply gender stratification, miR-223-3p and miR-379-5p were upregulated only in men. Therefore, miRNAs play essential role in maintaining stable MS course and this process has certain gender-specific differences.

Pharmacogenetics of glatiramer acetate therapy for multiple sclerosis: the impact of genome-wide association studies identified disease risk loci.

AIM: Association analysis of genome-wide association studies (GWAS) identified multiple sclerosis (MS) risk genetic variants with glatiramer acetate (GA) treatment efficacy. PATIENTS & METHODS: SNPs in 17 GWAS-identified immune response loci were analyzed in 296 Russian MS patients as possible markers of optimal GA treatment response for at least 2 years. RESULTS: Alleles/genotypes of EOMES, CLEC16A, IL22RA2, PVT1 and HLA-DRB1 were associated by themselves with event-free phenotype during GA treatment for at least 2 years (p f  = 0.032 - 0.00092). The biallelic combinations including EOMES, CLEC16A, IL22RA2, PVT1, TYK2, CD6, IL7RA and IRF8 genes were associated with response to GA with increased significance level (p f  = 0.0060 - 1.1 × 10-5). The epistasic interactions or additive effects were observed between the components of the identified biallelic combinations. CONCLUSION: We pinpointed the involvement of several GWAS-identified MS risk loci in GA therapy efficacy. These findings may be aggregated to predict the optimal GA response in MS patients.

Variants of MicroRNA Genes: Gender-Specific Associations with Multiple Sclerosis Risk and Severity.

Multiple sclerosis (MS) is an autoimmune neuro-inflammatory disease arising from complex interactions of genetic, epigenetic, and environmental factors. Variations in genes of some microRNAs--key post-transcriptional regulators of many genes--can influence microRNAs expression/function and contribute to MS via expression changes of protein-coding target mRNA genes. We performed an association study of polymorphous variants of MIR146A rs2910164, MIR196A2 rs11614913, MIR499A rs3746444 MIR223 rs1044165 and their combinations with MS risk and severity. 561 unrelated patients with bout-onset MS and 441 healthy volunteers were enrolled in the study. We observed associations of MS risk with allele MIR223*T and combination (MIR223*T + MIR146A*G/G) carriage in the entire groups and in women at Bonferroni-corrected significance level (pcorr < 0.05). Besides, MIR146A*G/G association with MS was observed in women with nominal significance (pf = 0.025). No MS associations were found in men. A more severe MS course (MSSS value > 3.5) was associated with the carriage of MIR499A*C/T and, less reliably, of MIR499A*C (pcorr = 0.006 and pcorr = 0.024, respectively) and with the carriage of combinations (MIR499A*C/T + MIR196A2*C) and (MIR499A*C + MIR196A2*C) (pcorr = 0.00078 and pcorr = 0.0059, respectively). These associations also showed gender specificity, as they were not significant in men and substantially reinforced in women. The strongest association with MS severity was observed in women for combination (MIR499A*C/T + MIR196A2*C): pcorr = 4.43 × 10(-6) and OR = 3.23 (CI: 1.99-5.26).