Applicability of Diagnostic Criteria and High Prevalence of Familial Dysbetalipoproteinemia in Russia: A Pilot Study.

Familial dysbetalipoproteinemia (FD) is a highly atherogenic genetically based lipid disorder with an underestimated actual prevalence. In recent years, several biochemical algorithms have been developed to diagnose FD using available laboratory tests. The practical applicability of FD diagnostic criteria and the prevalence of FD in Russia have not been previously assessed. We demonstrated that the diagnostic algorithms of FD, including the diagnostic apoB levels, require correction, taking into account the distribution of apoB levels in the population. At the same time, a triglycerides cutoff ≥ 1.5 mmol/L may be a useful tool in identifying subjects with FD. In this study, a high prevalence of FD was detected: 0.67% (one in 150) based on the ε2ε2 haplotype and triglycerides levels ≥ 1.5 mmol/L. We also analyzed the presence and pathogenicity of APOE variants associated with autosomal dominant FD in a large research sample.

Genetic landscape in Russian patients with familial left ventricular noncompaction.

Background: Left ventricular noncompaction (LVNC) cardiomyopathy is a disorder that can be complicated by heart failure, arrhythmias, thromboembolism, and sudden cardiac death. The aim of this study is to clarify the genetic landscape of LVNC in a large cohort of well-phenotyped Russian patients with LVNC, including 48 families (n=214). Methods: All index patients underwent clinical examination and genetic analysis, as well as family members who agreed to participate in the clinical study and/or in the genetic testing. The genetic testing included next generation sequencing and genetic classification according to ACMG guidelines. Results: A total of 55 alleles of 54 pathogenic and likely pathogenic variants in 24 genes were identified, with the largest number in the MYH7 and TTN genes. A significant proportion of variants -8 of 54 (14.8%) -have not been described earlier in other populations and may be specific to LVNC patients in Russia. In LVNC patients, the presence of each subsequent variant is associated with increased odds of having more severe LVNC subtypes than isolated LVNC with preserved ejection fraction. The corresponding odds ratio is 2.77 (1.37 -7.37; p <0.001) per variant after adjustment for sex, age, and family. Conclusion: Overall, the genetic analysis of LVNC patients, accompanied by cardiomyopathy-related family history analysis, resulted in a high diagnostic yield of 89.6%. These results suggest that genetic screening should be applied to the diagnosis and prognosis of LVNC patients.

The Metagenomic Analysis of Viral Diversity in Colorado Potato Beetle Public NGS Data.

The Colorado potato beetle (CPB) is one of the most serious insect pests due to its high ecological plasticity and ability to rapidly develop resistance to insecticides. The use of biological insecticides based on viruses is a promising approach to control insect pests, but the information on viruses which infect leaf feeding beetles is scarce. We performed a metagenomic analysis of 297 CPB genomic and transcriptomic samples from the public National Center for Biotechnology Information Sequence Read Archive (NCBI SRA) database. The reads that were not aligned to the reference genome were assembled with metaSPAdes, and 13314 selected contigs were analyzed with BLAST tools. The contigs and non-aligned reads were also analyzed with Kraken2 software. A total of 3137 virus-positive contigs were attributed to different viruses belonging to 6 types, 17 orders, and 32 families, matching over 97 viral species. The annotated sequences can be divided into several groups: those that are homologous to genetic sequences of insect viruses (Adintoviridae, Ascoviridae, Baculoviridae, Dicistroviridae, Chuviridae, Hytrosaviridae, Iflaviridae, Iridoviridae, Nimaviridae, Nudiviridae, Phasmaviridae, Picornaviridae, Polydnaviriformidae, Xinmoviridae etc.), plant viruses (Betaflexiviridae, Bromoviridae, Kitaviridae, Potyviridae), and endogenous retroviral elements (Retroviridae, Metaviridae). Additionally, the full-length genomes and near-full length genome sequences of several viruses were assembled. We also found sequences belonging to Bracoviriform viruses and, for the first time, experimentally validated the presence of bracoviral genetic fragments in the CPB genome. Our work represents the first attempt to discover the viral genetic material in CPB samples, and we hope that further studies will help to identify new viruses to extend the arsenal of biopesticides against CPB.

Identification of Pathogenic Variant Burden and Selection of Optimal Diagnostic Method Is a Way to Improve Carrier Screening for Autosomal Recessive Diseases.

Cystic fibrosis, phenylketonuria, alpha-1 antitrypsin deficiency, and sensorineural hearing loss are among the most common autosomal recessive diseases, which require carrier screening. The evaluation of population allele frequencies (AF) of pathogenic variants in genes associated with these conditions and the choice of the best genotyping method are the necessary steps toward development and practical implementation of carrier-screening programs. We performed custom panel genotyping of 3821 unrelated participants from two Russian population representative samples and three patient groups using real-time polymerase chain reaction (PCR) and next generation sequencing (NGS). The custom panel included 115 known pathogenic variants in the CFTR, PAH, SERPINA1, and GJB2 genes. Overall, 38 variants were detected. The comparison of genotyping platforms revealed the following advantages of real-time PCR: relatively low cost, simple genotyping data analysis, and easier detection of large indels, while NGS showed better accuracy of variants identification and capability for detection of additional pathogenic variants in adjacent regions. A total of 23 variants had significant differences in estimated AF comparing with non-Finnish Europeans from gnomAD. This study provides new AF data for variants associated with the studied disorders and the comparison of genotyping methods for carrier screening.

A Case of Severe Left-Ventricular Noncompaction Associated with Splicing Altering Variant in the FHOD3 Gene.

Left ventricular noncompaction (LVNC) is a highly heterogeneous primary disorder of the myocardium. Its clinical features and genetic spectrum strongly overlap with other types of primary cardiomyopathies, in particular, hypertrophic cardiomyopathy. Study and the accumulation of genotype-phenotype correlations are the way to improve the precision of our diagnostics. We present a familial case of LVNC with arrhythmic and thrombotic complications, myocardial fibrosis and heart failure, cosegregating with the splicing variant in the FHOD3 gene. This is the first description of FHOD3-dependent LVNC to our knowledge. We also revise the assumed mechanism of pathogenesis in the case of FHOD3 splicing alterations.

Generation of induced pluripotent stem cell line, ICGi034-A, by reprogramming peripheral blood mononuclear cells from a patient with Parkinson's disease associated with GBA mutation.

Mutation in the glucocerebrosidase encoding gene (GBA) is one of the most frequent genetic cause of Parkinson's disease. ICGi034-A induced pluripotent stem cell (iPSC) line obtained by reprogramming peripheral blood mononuclear cells (PBMCs) of a patient with heterozygous c.1226A > G (p.N370S) mutation in the GBA gene can be used for studying the fundamental mechanisms of the pathogenesis of GBA-associated Parkinson's disease, and for potential drug screening. The iPSCs express pluripotency markers (NANOG, SSEA4, TRA-1-60, OCT4, SOX2), have a normal karyotype, and are capable of producing derivatives of three germ layers.

Targeted Sequencing of 242 Clinically Important Genes in the Russian Population From the Ivanovo Region.

We performed a targeted sequencing of 242 clinically important genes mostly associated with cardiovascular diseases in a representative population sample of 1,658 individuals from the Ivanovo region northeast of Moscow. Approximately 11% of 11,876 detected variants were not found in the Single Nucleotide Polymorphism Database (dbSNP) or reported earlier in the Russian population. Most novel variants were singletons and doubletons in our sample, and virtually no novel alleles presumably specific for the Russian population were able to reach the frequencies above 0.1-0.2%. The overwhelming majority (99.3%) of variants detected in this study in three or more copies were shared with other populations. We found two dominant and seven recessive known pathogenic variants with allele frequencies significantly increased compared to those in the gnomAD non-Finnish Europeans. Of the 242 targeted genes, 28 were in the list of 59 genes for which the American College of Medical Genetics and Genomics (ACMG) recommended the reporting of incidental findings. Based on the number of variants detected in the sequenced subset of ACMG59 genes, we approximated the prevalence of known pathogenic and novel or rare protein-truncating variants in the complete set of ACMG59 genes in the Ivanovo population at 1.4 and 2.8%, respectively. We analyzed the available clinical data and observed the incomplete penetrance of known pathogenic variants in the 28 ACMG59 genes: only 1 individual out of 12 with such variants had the phenotype most likely related to the variant. When known pathogenic and novel or rare protein-truncating variants were considered together, the overall rate of confirmed phenotypes was about 19%, with maximum in the subset of novel protein-truncating variants. We report three novel protein truncating variants in APOB and one in MYH7 observed in individuals with hypobetalipoproteinemia and hypertrophic cardiomyopathy, respectively. Our results provide a valuable reference for the clinical interpretation of gene sequencing in Russian and other populations.

Very long intergenic non-coding (vlinc) RNAs directly regulate multiple genes in cis and trans.

BACKGROUND: The majority of the human genome is transcribed in the form of long non-coding (lnc) RNAs. While these transcripts have attracted considerable interest, their molecular mechanisms of function and biological significance remain controversial. One of the main reasons behind this lies in the significant challenges posed by lncRNAs requiring the development of novel methods and concepts to unravel their functionality. Existing methods often lack cross-validation and independent confirmation by different methodologies and therefore leave significant ambiguity as to the authenticity of the outcomes. Nonetheless, despite all the caveats, it appears that lncRNAs may function, at least in part, by regulating other genes via chromatin interactions. Therefore, the function of a lncRNA could be inferred from the function of genes it regulates. In this work, we present a genome-wide functional annotation strategy for lncRNAs based on identification of their regulatory networks via the integration of three distinct types of approaches: co-expression analysis, mapping of lncRNA-chromatin interactions, and assaying molecular effects of lncRNA knockdowns obtained using an inducible and highly specific CRISPR/Cas13 system. RESULTS: We applied the strategy to annotate 407 very long intergenic non-coding (vlinc) RNAs belonging to a novel widespread subclass of lncRNAs. We show that vlincRNAs indeed appear to regulate multiple genes encoding proteins predominantly involved in RNA- and development-related functions, cell cycle, and cellular adhesion via a mechanism involving proximity between vlincRNAs and their targets in the nucleus. A typical vlincRNAs can be both a positive and negative regulator and regulate multiple genes both in trans and cis. Finally, we show vlincRNAs and their regulatory networks potentially represent novel components of DNA damage response and are functionally important for the ability of cancer cells to survive genotoxic stress. CONCLUSIONS: This study provides strong evidence for the regulatory role of the vlincRNA class of lncRNAs and a potentially important role played by these transcripts in the hidden layer of RNA-based regulation in complex biological systems.

Style transfer with variational autoencoders is a promising approach to RNA-Seq data harmonization and analysis.

MOTIVATION: The transcriptomic data are being frequently used in the research of biomarker genes of different diseases and biological states. The most common tasks there are the data harmonization and treatment outcome prediction. Both of them can be addressed via the style transfer approach. Either technical factors or any biological details about the samples which we would like to control (gender, biological state, treatment, etc.) can be used as style components. RESULTS: The proposed style transfer solution is based on Conditional Variational Autoencoders, Y-Autoencoders and adversarial feature decomposition. To quantitatively measure the quality of the style transfer, neural network classifiers which predict the style and semantics after training on real expression were used. Comparison with several existing style-transfer based approaches shows that proposed model has the highest style prediction accuracy on all considered datasets while having comparable or the best semantics prediction accuracy. AVAILABILITY AND IMPLEMENTATION: https://github.com/NRshka/stvae-source. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Biomarker discovery in attention deficit hyperactivity disorder: RNA sequencing of whole blood in discordant twin and case-controlled cohorts.

BACKGROUND: A variety of DNA-based methods have been applied to identify genetic markers of attention deficit hyperactivity disorder (ADHD), but the connection to RNA-based gene expression has not been fully exploited. METHODS: Using well defined cohorts of discordant, monozygotic twins from the Michigan State University Twin Registry, and case-controlled ADHD cases in adolescents, the present studies utilized advanced single molecule RNA sequencing to identify expressed changes in whole blood RNA in ADHD. Multiple analytical strategies were employed to narrow differentially expressed RNA targets to a small set of potential biomarkers of ADHD. RESULTS: RNA markers common to both the discordant twin study and case-controlled subjects further narrowed the putative targets, some of which had been previously associated with ADHD at the DNA level. The potential role of several differentially expressed genes, including ABCB5, RGS2, GAK, GIT1 and 3 members of the galactose metabolism pathway (GALE, GALT, GALK1) are substantiated by prior associations to ADHD and by established mechanistic connections to molecular pathways relevant to ADHD and behavioral control. CONCLUSIONS: The convergence of DNA, RNA, and metabolic data suggests these may be promising targets for diagnostics and therapeutics in ADHD.

Identification of novel GLI1 target genes and regulatory circuits in human cancer cells.

Hedgehog (HH) signaling is involved in many physiological processes, and pathway deregulation can result in a wide range of malignancies. Glioma-associated oncogene 1 (GLI1) is a transcription factor and a terminal effector of the HH cascade. Despite its crucial role in tumorigenesis, our understanding of the GLI1 cellular targets is quite limited. In this study, we identified multiple new GLI1 target genes using a combination of different genomic surveys and then subjected them to in-depth validation in human cancer cell lines. We were able to validate >90% of the new targets, which were enriched in functions involved in neurogenesis and regulation of transcription, in at least one type of follow-up experiment. Strikingly, we found that RNA editing of GLI1 can modulate effects on the targets. Furthermore, one of the top targets, FOXS1, a gene encoding a transcription factor previously implicated in nervous system development, was shown to act in a negative feedback loop limiting the cellular effects of GLI1 in medulloblastoma and rhabdomyosarcoma cells. Moreover, FOXS1 is both highly expressed and positively correlated with GLI1 in medulloblastoma samples of the Sonic HH subgroup, further arguing for the existence of FOXS1/GLI1 interplay in human tumors. Consistently, high FOXS1 expression predicts longer relapse-free survival in breast cancer. Overall, our findings open multiple new avenues in HH signaling pathway research and have potential for translational implications.

Deep Sequencing Transcriptome Analysis of Murine Wound Healing: Effects of a Multicomponent, Multitarget Natural Product Therapy-Tr14.

Wound healing involves an orchestrated response that engages multiple processes, such as hemostasis, cellular migration, extracellular matrix synthesis, and in particular, inflammation. Using a murine model of cutaneous wound repair, the transcriptome was mapped from 12 h to 8 days post-injury, and in response to a multicomponent, multi-target natural product, Tr14. Using single-molecule RNA sequencing (RNA-seq), there were clear temporal changes in known transcripts related to wound healing pathways, and additional novel transcripts of both coding and non-coding genes. Tr14 treatment modulated >100 transcripts related to key wound repair pathways, such as response to wounding, wound contraction, and cytokine response. The results provide the most precise and comprehensive characterization to date of the transcriptome's response to skin damage, repair, and multicomponent natural product therapy. By understanding the wound repair process, and the effects of natural products, it should be possible to intervene more effectively in diseases involving aberrant repair.

Genome-wide profiling and differential expression of microRNA in rat pluripotent stem cells.

MicroRNAs (miRNAs) constitute a class of small noncoding RNAs that plays an important role in the post-transcriptional regulation of gene expression. Much evidence has demonstrated that miRNAs are involved in regulating the human and mouse pluripotency. Nevertheless, to our knowledge, miRNAs in the pluripotent stem cells of one of the most commonly used model organisms - the Rattus norvegicus have not been studied. In the present study, we performed deep sequencing of small RNA molecules in the embryonic fibroblasts, embryonic stem cells, and induced pluripotent stem cells of laboratory rats. Bioinformatics analysis revealed 674 known miRNAs and 394 novel miRNA candidates in all of the samples. Expression of known pluripotency-associated miRNAs, such as the miR-290-295 and miR-183-96-182 clusters as well as members of the miR-200 family, was detected in rat pluripotent stem cells. Analysis of the targets of differentially expressed known and novel miRNAs showed their involvement in the regulation of pluripotency and the reprogramming process in rats. Bioinformatics and systems biology approaches identified potential pathways that are regulated by these miRNAs. This study contributes to our understanding of miRNAs in the regulation of pluripotency and cell reprogramming in the laboratory rat.

Functional annotation of the vlinc class of non-coding RNAs using systems biology approach.

Functionality of the non-coding transcripts encoded by the human genome is the coveted goal of the modern genomics research. While commonly relied on the classical methods of forward genetics, integration of different genomics datasets in a global Systems Biology fashion presents a more productive avenue of achieving this very complex aim. Here we report application of a Systems Biology-based approach to dissect functionality of a newly identified vast class of very long intergenic non-coding (vlinc) RNAs. Using highly quantitative FANTOM5 CAGE dataset, we show that these RNAs could be grouped into 1542 novel human genes based on analysis of insulators that we show here indeed function as genomic barrier elements. We show that vlinc RNAs genes likely function in cisto activate nearby genes. This effect while most pronounced in closely spaced vlinc RNA-gene pairs can be detected over relatively large genomic distances. Furthermore, we identified 101 vlinc RNA genes likely involved in early embryogenesis based on patterns of their expression and regulation. We also found another 109 such genes potentially involved in cellular functions also happening at early stages of development such as proliferation, migration and apoptosis. Overall, we show that Systems Biology-based methods have great promise for functional annotation of non-coding RNAs.

Dark matter RNA illuminates the puzzle of genome-wide association studies.

In the past decade, numerous studies have made connections between sequence variants in human genomes and predisposition to complex diseases. However, most of these variants lie outside of the charted regions of the human genome whose function we understand; that is, the sequences that encode proteins. Consequently, the general concept of a mechanism that translates these variants into predisposition to diseases has been lacking, potentially calling into question the validity of these studies. Here we make a connection between the growing class of apparently functional RNAs that do not encode proteins and whose function we do not yet understand (the so-called 'dark matter' RNAs) and the disease-associated variants. We review advances made in a different genomic mapping effort - unbiased profiling of all RNA transcribed from the human genome - and provide arguments that the disease-associated variants exert their effects via perturbation of regulatory properties of non-coding RNAs existing in mammalian cells.

On the importance of small changes in RNA expression.

The analysis of the differential expression of genes has been the key goal of many molecular biology methods for decades and will remain with us for decades to come. It constitutes a fundamental resource at our disposal for determining the relationship between products of transcription, biology and disease. The completed genome sequencing of many common species allowed microarrays and RNA sequencing (RNAseq) to become major tools in Systems Biology. However, we estimate that at least half of all experiments ignore transcripts that change less than some subjectively chosen threshold, typically around 2-3 fold. Here we show that a majority of the informative RNAs and differentially expressed transcripts can exhibit fold changes less than 2. We use highly quantitative single-molecule sequencing of total cellular RNA derived from a time course of inflammatory response, a process critical to a large number of diseases. Furthermore, we show that enrichment of biologically-relevant functions occurs even at very low fold changes in RNA levels. In addition, we show that most of the common statistical methods can reliably detect transcripts with low fold change when as few as 3 biological replicates are sequenced using single-molecule based RNAseq. In conclusion, given the prevalence of expression profiling in current research, the loss of data in half of all expression studies results in a significant, yet needless drain on the discovery process.