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.

Lung inflammation is associated with lipid deposition.

Lung inflammation, pneumonia, is an acute respiratory disease of varying etiology that has recently drawn much attention during the COVID-19 pandemic as lungs are among the main targets for SARS-CoV-2. Multiple other etiological agents are associated with pneumonias. Here, we describe a newly-recognized pathology, namely abnormal lipid depositions in the lungs of patients who died from COVID-19 as well as from non-COVID-19 pneumonias. Our analysis of both semi-thin and Sudan III-stained lung specimens revealed extracellular and intracellular lipid depositions irrespective of the pneumonia etiology. Most notably, lipid depositions were located within vessels adjacent to inflamed regions, where they apparently interfere with the blood flow. Structurally, the lipid droplets in the inflamed lung tissue were homogeneous and lacked outer membranes as assessed by electron microscopy. Morphometric analysis of lipid droplet deposition area allowed us to distinguish the non-pneumonia control lung specimens from the macroscopically intact area of the pneumonia lung and from the inflamed area of the pneumonia lung. Our measurements revealed a gradient of lipid deposition towards the inflamed region. The pattern of lipid distribution proved universal for all pneumonias. Finally, lipid metabolism in the lung tissue was assessed by the fatty acid analysis and by expression of genes involved in lipid turnover. Chromato-mass spectrometry revealed that unsaturated fatty acid content was elevated at inflammation sites compared to that in control non-inflamed lung tissue from the same individual. The expression of genes involved in lipid metabolism was altered in pneumonia, as shown by qPCR and in silico RNA-seq analysis. Thus, pneumonias of various etiologies are associated with specific lipid abnormalities; therefore, lipid metabolism can be considered to be a target for new therapeutic strategies.

SARS-CoV-2 cellular tropism and direct multiorgan failure in COVID-19 patients: Bioinformatic predictions, experimental observations, and open questions.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19), has led to an unprecedented public health emergency worldwide. While common cold symptoms are observed in mild cases, COVID-19 is accompanied by multiorgan failure in severe patients. Organ damage in COVID-19 patients is partially associated with the indirect effects of SARS-CoV-2 infection (e.g., systemic inflammation, hypoxic-ischemic damage, coagulopathy), but early processes in COVID-19 patients that trigger a chain of indirect effects are connected with the direct infection of cells by the virus. To understand the virus transmission routes and the reasons for the wide-spectrum of complications and severe outcomes of COVID-19, it is important to identify the cells targeted by SARS-CoV-2. This review summarizes the major steps of investigation and the most recent findings regarding SARS-CoV-2 cellular tropism and the possible connection between the early stages of infection and multiorgan failure in COVID-19. The SARS-CoV-2 pandemic is the first epidemic in which data extracted from single-cell RNA-seq (scRNA-seq) gene expression data sets have been widely used to predict cellular tropism. The analysis presented here indicates that the SARS-CoV-2 cellular tropism predictions are accurate enough for estimating the potential susceptibility of different cells to SARS-CoV-2 infection; however, it appears that not all susceptible cells may be infected in patients with COVID-19.

Intragenic compensation through the lens of deep mutational scanning.

A significant fraction of mutations in proteins are deleterious and result in adverse consequences for protein function, stability, or interaction with other molecules. Intragenic compensation is a specific case of positive epistasis when a neutral missense mutation cancels effect of a deleterious mutation in the same protein. Permissive compensatory mutations facilitate protein evolution, since without them all sequences would be extremely conserved. Understanding compensatory mechanisms is an important scientific challenge at the intersection of protein biophysics and evolution. In human genetics, intragenic compensatory interactions are important since they may result in variable penetrance of pathogenic mutations or fixation of pathogenic human alleles in orthologous proteins from related species. The latter phenomenon complicates computational and clinical inference of an allele's pathogenicity. Deep mutational scanning is a relatively new technique that enables experimental studies of functional effects of thousands of mutations in proteins. We review the important aspects of the field and discuss existing limitations of current datasets. We reviewed ten published DMS datasets with quantified functional effects of single and double mutations and described rates and patterns of intragenic compensation in eight of them. Supplementary Information: The online version contains supplementary material available at 10.1007/s12551-022-01005-w.

Ectopic expression of HIV-1 Tat modifies gene expression in cultured B cells: implications for the development of B-cell lymphomas in HIV-1-infected patients.

An increased frequency of B-cell lymphomas is observed in human immunodeficiency virus-1 (HIV-1)-infected patients, although HIV-1 does not infect B cells. Development of B-cell lymphomas may be potentially due to the action of the HIV-1 Tat protein, which is actively released from HIV-1-infected cells, on uninfected B cells. The exact mechanism of Tat-induced B-cell lymphomagenesis has not yet been precisely identified. Here, we ectopically expressed either Tat or its TatC22G mutant devoid of transactivation activity in the RPMI 8866 lymphoblastoid B cell line and performed a genome-wide analysis of host gene expression. Stable expression of both Tat and TatC22G led to substantial modifications of the host transcriptome, including pronounced changes in antiviral response and cell cycle pathways. We did not find any strong action of Tat on cell proliferation, but during prolonged culturing, Tat-expressing cells were displaced by non-expressing cells, indicating that Tat expression slightly inhibited cell growth. We also found an increased frequency of chromosome aberrations in cells expressing Tat. Thus, Tat can modify gene expression in cultured B cells, leading to subtle modifications in cellular growth and chromosome instability, which could promote lymphomagenesis over time.

Mesenchymal stromal cell-derived extracellular vesicles afford neuroprotection by modulating PI3K/AKT pathway and calcium oscillations.

Mesenchymal stromal cells (MSC) are widely recognized as potential effectors in neuroprotective therapy. The protective properties of MSC were considered to be associated with the secretion of extracellular vesicles (MSC-EV). We explored the effects of MSC-EV in vivo on models of traumatic and hypoxia-ischemia (HI) brain injury. Neuroprotective mechanisms triggered by MSC-EV were also studied in vitro using a primary neuroglial culture. Intranasal administration of MSC-EV reduced the volume of traumatic brain damage, correlating with a recovery of sensorimotor functions. Neonatal HI-induced brain damage was mitigated by the MSC-EV administration. This therapy also promoted the recovery of sensorimotor functions, implying enhanced neuroplasticity, and MSC-EV-induced growth of neurites in vitro supports this. In the in vitro ischemic model, MSC-EV prevented cell calcium (Ca2+) overload and subsequent cell death. In mixed neuroglial culture, MSC-EV induced inositol trisphosphate (IP3) receptor-related Ca2+ oscillations in astrocytes were associated with resistance to calcium overload not only in astrocytes but also in co-cultured neurons, demonstrating intercellular positive crosstalk between neural cells. This implies that phosphatidylinositol 3-Kinase/AKT signaling is one of the main pathways in MSC-EV-mediated protection of neural cells exposed to ischemic challenge. Components of this pathway were identified among the most enriched categories in the MSC-EV proteome.

Phenotypic vs. genetic cascade screening for familial hypercholesterolemia: A case report.

One of the most common autosomal dominant disorders is familial hypercholesterolemia (FH), causing premature atherosclerotic cardiovascular diseases and a high risk of death due to lifelong exposure to elevated low-density lipoprotein cholesterol (LDL-C) levels. FH has a proven arsenal of treatments and the opportunity for genetic diagnosis. Despite this, FH remains largely underdiagnosed worldwide. Cascade screening is a cost-effective method for the identification of new patients with FH and the prevention of cardiovascular diseases. It is usually based only on clinical data. We describe a 48-year-old index patient with a very high LDL-C level without controlled guidelines-based medication, premature atherosclerosis, and a rare variant in the low-density lipoprotein receptor (LDLR) gene. Phenotypic cascade screening identified three additional FH relatives, namely the proband's daughter, and two young grandsons. The genetic screening made it possible to rule out FH in the proband's younger grandson. This clinical case demonstrates that genetic cascade screening is the most effective way of identifying new FH cases. We also first described in detail the phenotype of patients with a likely pathogenic variant LDLR-p.K223_D227dup.

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.

Do Extracellular Vesicles Derived from Mesenchymal Stem Cells Contain Functional Mitochondria?

Extracellular vesicles (EV) derived from stem cells have become an effective complement to the use in cell therapy of stem cells themselves, which has led to an explosion of research into the mechanisms of vesicle formation and their action. There is evidence demonstrating the presence of mitochondrial components in EV, but a definitive conclusion about whether EV contains fully functional mitochondria has not yet been made. In this study, two EV fractions derived from mesenchymal stromal stem cells (MSC) and separated by their size were examined. Flow cytometry revealed the presence of mitochondrial lipid components capable of interacting with mitochondrial dyes MitoTracker Green and 10-nonylacridine orange; however, the EV response to the probe for mitochondrial membrane potential was negative. Detailed analysis revealed components from all mitochondria compartments, including house-keeping mitochondria proteins and DNA as well as energy-related proteins such as membrane-localized proteins of complexes I, IV, and V, and soluble proteins from the Krebs cycle. When assessing the functional activity of mitochondria, high variability in oxygen consumption was noted, which was only partially attributed to mitochondrial respiratory activity. Our findings demonstrate that the EV contain all parts of mitochondria; however, their independent functionality inside EV has not been confirmed, which may be due either to the absence of necessary cofactors and/or the EV formation process and, probably the methodology of obtaining EV.

OrthoQuantum: visualizing evolutionary repertoire of eukaryotic proteins.

Extensive amounts of data from next-generation sequencing and omics studies have led to the accumulation of information that provides insight into the evolutionary landscape of related proteins. Here, we present OrthoQuantum, a web server that allows for time-efficient analysis and visualization of phylogenetic profiles of any set of eukaryotic proteins. It is a simple-to-use tool capable of searching large input sets of proteins. Using data from open source databases of orthologous sequences in a wide range of taxonomic groups, it enables users to assess coupled evolutionary patterns and helps define lineage-specific innovations. The web interface allows to perform queries with gene names and UniProt identifiers in different phylogenetic clades and supplement presence with an additional BLAST search. The conservation patterns of proteins are coded as binary vectors, i.e., strings that encode the presence or absence of orthologous proteins in other genomes. These strings are used to calculate top-scoring correlation pairs needed for finding co-inherited proteins which are simultaneously present or simultaneously absent in specific lineages. Profiles are visualized in combination with phylogenetic trees in a JavaScript-based interface. The OrthoQuantum v1.0 web server is freely available at http://orthoq.bioinf.fbb.msu.ru along with documentation and tutorial.

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.

Gut Microbiota as a Source of Uremic Toxins.

Uremic retention solutes are the compounds that accumulate in the blood when kidney excretory function is impaired. Some of these compounds are toxic at high concentrations and are usually known as "uremic toxins". The cumulative detrimental effect of uremic toxins results in numerous health problems and eventually mortality during acute or chronic uremia, especially in end-stage renal disease. More than 100 different solutes increase during uremia; however, the exact origin for most of them is still debatable. There are three main sources for such compounds: exogenous ones are consumed with food, whereas endogenous ones are produced by the host metabolism or by symbiotic microbiota metabolism. In this article, we identify uremic retention solutes presumably of gut microbiota origin. We used database analysis to obtain data on the enzymatic reactions in bacteria and human organisms that potentially yield uremic retention solutes and hence to determine what toxins could be synthesized in bacteria residing in the human gut. We selected biochemical pathways resulting in uremic retention solutes synthesis related to specific bacterial strains and revealed links between toxin concentration in uremia and the proportion of different bacteria species which can synthesize the toxin. The detected bacterial species essential for the synthesis of uremic retention solutes were then verified using the Human Microbiome Project database. Moreover, we defined the relative abundance of human toxin-generating enzymes as well as the possibility of the synthesis of a particular toxin by the human metabolism. Our study presents a novel bioinformatics approach for the elucidation of the origin of both uremic retention solutes and uremic toxins and for searching for the most likely human microbiome producers of toxins that can be targeted and used for the therapy of adverse consequences of uremia.

Personalized approach in arrhythmology by genetic-based data: a case report.

The results of molecular genetic testing may affect recommended treatment or therapeutic decisions and risk assessment, may help with identification of family members at risk. Here, we report a case of a young patient with a paradoxical combination of two inherited arrhythmic syndromes and demonstrate the role of genetic testing as one of the basis of personalized approach in diagnosis, treatment and prevention complications of inherited channelopathies complications. Integration of genetic testing results into clinical practice is a successful example of the concept of personalized medicine.

The results of genetic testing may help to clarify the diagnosis, help the doctor to choose treatment and patient management tactics. We report a case of a young patient with the relatively rare arrythmia. We are highlighting the role of genetic testing as a basis of personalized approach of arrhythmia patient.

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.

The Double Mutation DSG2-p.S363X and TBX20-p.D278X Is Associated with Left Ventricular Non-Compaction Cardiomyopathy: Case Report.

Left ventricular non-compaction cardiomyopathy (LVNC) is a rare heart disease, with or without left ventricular dysfunction, which is characterized by a two-layer structure of the myocardium and an increased number of trabeculae. The study of familial forms of LVNC is helpful for risk prediction and genetic counseling of relatives. Here, we present a family consisting of three members with LVNC. Using a next-generation sequencing approach a combination of two (likely) pathogenic nonsense mutations DSG2-p.S363X and TBX20-p.D278X was identified in all three patients. TBX20 encodes the cardiac T-box transcription factor 20. DSG2 encodes desmoglein-2, which is part of the cardiac desmosomes and belongs to the cadherin family. Since the identified nonsense variant (DSG2-p.S363X) is localized in the extracellular domain of DSG2, we performed in vitro cell transfection experiments. These experiments revealed the absence of truncated DSG2 at the plasma membrane, supporting the pathogenic relevance of DSG2-p.S363X. In conclusion, we suggest that in the future, these findings might be helpful for genetic screening and counseling of patients with LVNC.

Hemi- and Homozygous Loss-of-Function Mutations in DSG2 (Desmoglein-2) Cause Recessive Arrhythmogenic Cardiomyopathy with an Early Onset.

About 50% of patients with arrhythmogenic cardiomyopathy (ACM) carry a pathogenic or likely pathogenic mutation in the desmosomal genes. However, there is a significant number of patients without positive familial anamnesis. Therefore, the molecular reasons for ACM in these patients are frequently unknown and a genetic contribution might be underestimated. Here, we used a next-generation sequencing (NGS) approach and in addition single nucleotide polymor-phism (SNP) arrays for the genetic analysis of two independent index patients without familial medical history. Of note, this genetic strategy revealed a homozygous splice site mutation (DSG2-c.378+1G>T) in the first patient and a nonsense mutation (DSG2-p.L772X) in combination with a large deletion in DSG2 in the second one. In conclusion, a recessive inheritance pattern is likely for both cases, which might contribute to the hidden medical history in both families. This is the first report about these novel loss-of-function mutations in DSG2 that have not been previously identi-fied. Therefore, we suggest performing deep genetic analyses using NGS in combination with SNP arrays also for ACM index patients without obvious familial medical history. In the future, this finding might has relevance for the genetic counseling of similar cases.

The LDLR, APOB, and PCSK9 Variants of Index Patients with Familial Hypercholesterolemia in Russia.

Familial hypercholesterolemia (FH) is a common autosomal codominant disorder, characterized by elevated low-density lipoprotein cholesterol levels causing premature atherosclerotic cardiovascular disease. About 2900 variants of LDLR, APOB, and PCSK9 genes potentially associated with FH have been described earlier. Nevertheless, the genetics of FH in a Russian population is poorly understood. The aim of this study is to present data on the spectrum of LDLR, APOB, and PCSK9 gene variants in a cohort of 595 index Russian patients with FH, as well as an additional systematic analysis of the literature for the period of 1995-2020 on LDLR, APOB and PCSK9 gene variants described in Russian patients with FH. We used targeted and whole genome sequencing to search for variants. Accordingly, when combining our novel data and the data of a systematic literature review, we described 224 variants: 187 variants in LDLR, 14 variants in APOB, and 23 variants in PCSK9. A significant proportion of variants, 81 of 224 (36.1%), were not described earlier in FH patients in other populations and may be specific for Russia. Thus, this study significantly supplements knowledge about the spectrum of variants causing FH in Russia and may contribute to a wider implementation of genetic diagnostics in FH patients in Russia.

Differential Analysis of A-to-I mRNA Edited Sites in Parkinson's Disease.

Parkinson's disease (PD) is a widespread neuronal degenerative disorder with unexplored etiology. It is associated with various pathological events. In particular, the prefrontal cortex Brodmann area 9 (BA9) region is affected in PD. This frontal lobe brain region plays an important role in cognitive, motor, and memory-related functions. BA9 develops Lewy bodies in PD patients and shows essential changes in transcriptome and proteome, connected with mitochondria related pathways, protein folding pathways, and metallothioneins. Recently, altered adenosine to inosine mRNA editing patterns have been detected in various neurological pathologies. In this article, we present an investigation of differences in A-to-I RNA editing levels and specificity of mRNA editing sites in brain tissues of healthy and PD patients based on RNA sequencing data. Overall, decreased editing levels in the brains of PD patients were observed, potential editing sites with altered editing during PD were identified, and the role of different adenosine deaminases in this process was analyzed.

Expression of SARS-CoV-2 entry factors in lung epithelial stem cells and its potential implications for COVID-19.

SARS-CoV-2 can infiltrate the lower respiratory tract, resulting in severe respiratory failure and a high death rate. Normally, the airway and alveolar epithelium can be rapidly reconstituted by multipotent stem cells after episodes of infection. Here, we analyzed published RNA-seq datasets and demonstrated that cells of four different lung epithelial stem cell types express SARS-CoV-2 entry factors, including Ace2. Thus, stem cells can be potentially infected by SARS-CoV-2, which may lead to defects in regeneration capacity partially accounting for the severity of SARS-CoV-2 infection and its consequences.