Exome Sequencing for the Diagnostics of Osteogenesis Imperfecta in Six Russian Patients.

Osteogenesis imperfecta (OI) is a group of inherited disorders of connective tissue that cause significant deformities and fragility in bones. Most cases of OI are associated with pathogenic variants in collagen type I genes and are characterized by pronounced polymorphisms in clinical manifestations and the absence of clear phenotype-genotype correlation. The objective of this study was to conduct a comprehensive molecular-genetic and clinical analysis to verify the diagnosis of OI in six Russian patients with genetic variants in the COL1A1 and COL1A2 genes. Clinical and laboratory data were obtained from six OI patients who were observed at the Medical Genetics Center in Saint Petersburg from 2016 to 2023. Next-generation sequencing on MGISEQ G400 (MGI, China) was used for DNA analysis. The GATK bioinformatic software (version 4.5.0.0) was used for variant calling and hard filtering. Genetic variants were verified by the direct automatic sequencing of PCR products using the ABI 3500X sequencer. We identified six genetic variants, as follows pathogenic c.3505G>A (p. Gly1169Ser), c.769G>A (p.Gly257Arg), VUS c.4123G>A (p.Ala1375Thr), and c.4114A>T (p.Asn1372Tyr) in COL1A1; and likely pathogenic c.2035G>A (p.Gly679Ser) and c.739-2A>T in COL1A2. In addition, clinical cases are presented due to the presence of the c.4114A>T variant in the COL1A2 gene. Molecular genetics is essential for determining different OI types due to the high similarity across various types of the disease and the failure of unambiguous diagnosis based on clinical manifestations alone. Considering the variable approaches to OI classification, an integrated strategy is required for optimal patient management.

Exome sequencing in extreme altitude mountaineers identifies pathogenic variants in RTEL1 and COL6A1 previously associated with respiratory failure.

Adaptation of humans to challenging environmental conditions, such as extreme temperature, malnutrition, or hypoxia, is an interesting phenomenon for both basic and applied research. Identification of the genetic factors contributing to human adaptation to these conditions enhances our understanding of the underlying molecular and physiological mechanisms. In our study, we analyzed the exomes of 22 high altitude mountaineers to uncover genetic variants contributing to hypoxic adaptation. To our surprise, we identified two putative loss-of-function variants, rs1385101139 in RTEL1 and rs1002726737 in COL6A1 in two extremely high altitude (personal record of more than 8500 m) professional climbers. Both variants can be interpreted as pathogenic according to medical geneticists' guidelines, and are linked to inherited conditions involving respiratory failure (late-onset pulmonary fibrosis and severe Ullrich muscular dystrophy for rs1385101139 and rs1002726737, respectively). Our results suggest that a loss of gene function may act as an important factor of human adaptation, which is corroborated by previous reports in other human subjects.

A New Leu714Arg Variant in the Converter Domain of MYH7 is Associated with a Severe Form of Familial Hypertrophic Cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy is the most frequent autosomal dominant disease, yet due to genetic heterogeneity, incomplete penetrance, and phenotype variability, the prognosis of the disease course in pathogenic variant carriers remains an issue. Identifying common patterns among the effects of different genetic variants is important. METHODS: We investigated the cause of familial hypertrophic cardiomyopathy (HCM) in a family with two patients suffering from a particularly severe disease. Searching for the genetic variants in HCM genes was performed using different sequencing methods. RESULTS: A new missense variant, p.Leu714Arg, was identified in exon 19 of the beta-myosin heavy chain gene (MYH7). The mutation was found in a region that encodes the 'converter domain' in the globular myosin head. This domain is essential for the conformational change of myosin during ATP cleavage and contraction cycle. Most reports on different mutations in this region describe severe phenotypic consequences. The two patients with the p.Leu714Arg mutation had heart failure early in life and died from HCM complications. CONCLUSIONS: This case presents a new likely pathogenic variant in MYH7 and supports the hypothesis that myosin converter mutations constitute a subclass of HCM mutations with a poor prognosis for the patient.

Next-Generation Sequencing-Based Monitoring of Intestinal Bacteria and Bacteriophages Following Fecal Microbiota Transplantation in Inflammatory Bowel Diseases.

Inflammatory bowel diseases (IBD) and acute graft-versus-host disease (GVHD) are associated with persistent intestinal dysfunction preceded by gut bacterial dysbiosis. There are limited data on intestinal bacteriophages in these conditions. The aim of the present work was to detect associations between dominant intestinal bacteria by means of 16S rRNA gene sequencing, and some clinically significant viruses detected with a customized primer panel for NGS-based study. The clinical group included patients with Crohn's disease (IBD, n = 9), or GVHD (n = 6) subjected to fecal microbiota transplantation (FMT) from healthy donors. The stool specimens were taken initially, and 5 times post-FMT until day 120. Using NGS approach, we have found a higher abundance of Proteobacterota phylum in GVHD, especially, at later terms post-FMT. Moreover, we found an early increase of Klebsiella and E. coli/Shigella abundance in GVHD, along with decreased relative content of Faecalibacterium. Upon evaluation of intestinal phageome, the relative amount of Caudoviricetes class was higher in GVHD. A significant correlation was found between Proteobacteria and Caudoviricetes, thus suggesting their association during the post-FMT period. Moreover, the relative amounts of five Caudoviricetes phage species showed distinct correlations with Klebsiella and Enterococcus ratios at different terms of FMT. In conclusion, parallel use of 16S rRNA gene sequencing and targeted NGS viral panel is a feasible and useful option for tracing specific viral strains in fecal microbiota. The developed array of viral primers may be extended to detect other phages infecting the clinically relevant bacteria.

Human Exome Sequencing and Prospects for Predictive Medicine: Analysis of International Data and Own Experience.

Today, whole-exome sequencing (WES) is used to conduct the massive screening of structural and regulatory genes in order to identify the allele frequencies of disease-associated polymorphisms in various populations and thus detect pathogenic genetic changes (mutations or polymorphisms) conducive to malfunctional protein sequences. With its extensive capabilities, exome sequencing today allows both the diagnosis of monogenic diseases (MDs) and the examination of seemingly healthy populations to reveal a wide range of potential risks prior to disease manifestation (in the future, exome sequencing may outpace costly and less informative genome sequencing to become the first-line examination technique). This review establishes the human genetic passport as a new WES-based clinical concept for the identification of new candidate genes, gene variants, and molecular mechanisms in the diagnosis, prediction, and treatment of monogenic, oligogenic, and multifactorial diseases. Various diseases are addressed to demonstrate the extensive potential of WES and consider its advantages as well as disadvantages. Thus, WES can become a general test with a broad spectrum pf applications, including opportunistic screening.

Evaluation of Gut Microbiota in Healthy Persons and Type 1 Diabetes Mellitus Patients in North-Western Russia.

Bacterial microbiota in stool may vary over a wide range, depending on age, nutrition, etc. The purpose of our work was to discriminate phyla and genera of intestinal bacteria and their biodiversity within a healthy population (North-Western Russia) compared to the patients with type 1 diabetes mellitus (T1DM). The study group included 183 healthy persons 2 to 53 years old (a mean of 26.5±1.0 years old), and 41 T1DM patients (mean age 18.2±1.8 years old). The disease onset was at 11±1.5 years, with a T1DM experience of 7±1.5 years. Total DNA was isolated from the stool samples, and sequencing libraries were prepared by amplifying the V3-V4 region of the 16S rRNA gene sequenced by Illumina MiSeq. Bioinformatic processing of NGS databases was adapted for microbiota evalutaion. Despite the broad scatter, the biological diversity for bacterial microbiota expressed as the Shannon index was significantly increased from younger to older ages in the comparison group, higher in adult healthy persons, with a trend for decrease in the Actinomycetota phylum which includes Bifidobacterium longum species. Similar but non-significant age trends were noted in the T1DM group. Concordant with the Bacillota prevalence in stool samples of diabetic patients, some anaerobic bacteria (Faecalibacteria, Lachnospira and Ruminococcae, Roseburia) were enriched in the T1DM microbiome against controls. Hence, correction of microbiota for Ruminococcus and Lachnospiraceae requires future search for new probiotics. Lower abundance of Actinomycetota and Bifidobacter in T1DM suggests potential usage of Bifidobacter-based probiotics in this cohort.

The Telomeric Repeats of HHV-6A Do Not Determine the Chromosome into Which the Virus Is Integrated.

Human herpes virus 6A (HHV-6A) is able to integrate into the telomeric and subtelomeric regions of human chromosomes representing chromosomally integrated HHV-6A (ciHHV-6A). The integration starts from the right direct repeat (DRR) region. It has been shown experimentally that perfect telomeric repeats (pTMR) in the DRR region are required for the integration, while the absence of the imperfect telomeric repeats (impTMR) only slightly reduces the frequency of HHV-6 integration cases. The aim of this study was to determine whether telomeric repeats within DRR may define the chromosome into which the HHV-6A integrates. We analysed 66 HHV-6A genomes obtained from public databases. Insertion and deletion patterns of DRR regions were examined. We also compared TMR within the herpes virus DRR and human chromosome sequences retrieved from the Telomere-to-Telomere consortium. Our results show that telomeric repeats in DRR in circulating and ciHHV-6A have an affinity for all human chromosomes studied and thus do not define a chromosome for integration.

Russian Regional Differences in Allele Frequencies of CFTR Gene Variants: Genetic Monitoring of Infertile Couples.

A male factor, commonly associated with poor semen quality, is revealed in about 50% of infertile couples. CFTR gene (Cystic Fibrosis Transmembrane Conduction Regulator) variants are one of the common genetic causes of azoospermia-related male infertility. Notably, the spectrum and frequency of pathogenic CFTR variants vary between populations and geographical regions. In this work, we made an attempt to evaluate the allele frequency (AF) of 12 common CFTR variants in infertile Russian men and healthy individuals from different districts of Russia. Because of the limited number of population-based studies on Russian individuals, we characterized the population AFs based on data from the Registry of Russian cystic fibrosis (CF) patients. In addition to the CF patient registry, we estimated the local frequencies of the same set of variants based on the results of genotyping of CF patients in local biocollections (from St. Petersburg and Yugra regions). AFs of common CFTR variants calculated based on registry and biocollection data showed good concordance with directly measured population AFs. The estimated region-specific frequencies of CFTR variants allowed us to uncover statistically significant regional differences in the frequencies of the F508del (c.1521_1523del; p.Phe508del) and CFTRdele2,3(21kb) (c.54-5940_273+10250del21kb; p.Ser18ArgfsX) variants. The data from population-based studies confirmed previous observations that F508del, CFTRdele2,3(21kb), and L138ins (c.413_415dup; p.Leu138dup)variants are the most abundant among infertile patients, and their frequencies are significantly lower in healthy individuals and should be taken into account during genetic monitoring of the reproductive health of Russian individuals.

Clinical and Genetic Characteristics of Pediatric Patients with Hypophosphatasia in the Russian Population.

(1) Hypophosphatasia (HPP) is a rare inherited disease caused by mutations (pathogenic variants) in the ALPL gene which encodes tissue-nonspecific alkaline phosphatase (TNSALP). HPP is characterized by impaired bone mineral metabolism due to the low enzymatic activity of TNSALP. Knowledge about the structure of the gene and the features and functions of various ALPL gene variants, taking into account population specificity, gives an understanding of the hereditary nature of the disease, and contributes to the diagnosis, prevention, and treatment of the disease. The purpose of the study was to describe the spectrum and analyze the functional features of the ALPL gene variants, considering various HPP subtypes and clinical symptoms in Russian children. (2) From 2014−2021, the study included the blood samples obtained from 1612 patients with reduced alkaline phosphatase activity. The patients underwent an examination with an assessment of their clinical symptoms and biochemical levels of TNSALP. DNA was isolated from dried blood spots (DBSs) or blood from the patients to search for mutations in the exons of the ALPL gene using Sanger sequencing. The PCR products were sequenced using a reagent BigDye Terminator 3.1 kit (Applied Biosystems). Statistical analysis was performed using the GraphPad Prism 8.01 software. (3) The most common clinical symptoms in Russian patients with HPP and two of its variants (n = 22) were bone disorders (75%), hypomyotonia (50%), and respiratory failure (50%). The heterozygous carriage of the causal variants of the ALPL gene was detected in 225 patients. A total of 2 variants were found in 27 patients. In this group (n = 27), we identified 28 unique variants of the ALPL gene, of which 75.0% were missense, 17.9% were frameshift, 3.6% were splicing variants, and 3.6% were duplications. A total of 39.3% (11/28) of the variants were pathogenic, with two variants being probably pathogenic, and 15 variants had unknown clinical significance (VUS). Among the VUS group, 28.6% of the variants (7/28) were discovered by us for the first time. The most common variants were c.571G > A (p.Glu191Lys) and c.1171del (Arg391Valfs*12), with frequencies of 48.2% (13/28) and 11% (3/28), respectively. It was found that the frequency of nonsense variants of the ALPL gene was higher (p < 0.0001) in patients with the perinatal form compared to the infantile and childhood forms of HPP. Additionally, the number of homozygotes in patients with the perinatal form exceeded (p < 0.01) the frequencies of these genotypes in children with infantile and childhood forms of HPP. On the contrary, the frequencies of the compound-heterozygous and heterozygous genotypes were higher (p < 0.01) in patients with infantile childhood HPP than in perinatal HPP. In the perinatal form, residual TNSALP activity was lower (p < 0.0005) in comparison to the infantile and childhood (p < 0.05) forms of HPP. At the same time, patients with the heterozygous and compound-heterozygous genotypes (mainly missense variants) of the ALPL gene had greater residual activity (of the TNSALP protein) regarding those homozygous patients who were carriers of the nonsense variants (deletions and duplications) of the ALPL gene. Residual TNSALP activity was lower (p < 0.0001) in patients with pathogenic variants encoding the amino acids from the active site and the calcium and crown domains in comparison with the nonspecific region of the protein.

Description of the First Registered Case of Lopes-Maciel-Rodan Syndrome in Russia.

Lopes−Maciel−Rodan syndrome (LOMARS) is an extremely rare disorder, with only a few cases reported worldwide. LOMARS is caused by a compound heterozygous mutation in the HTT gene. Little is known about LOMARS pathogenesis and clinical manifestations. Whole exome sequencing (WES) was performed to achieve a definitive molecular diagnosis of the disorder. All NGS-identified variants underwent the Sanger confirmation. In addition, a literature review on genetic variations in the HTT gene was conducted. The paper reports a case of LOMARS in a pediatric patient in Russia. A preterm girl of non-consanguineous parents demonstrated severe psychomotor developmental delays in her first 12 months. By the age of 6 years, she failed to develop speech but was able to understand everyday phrases and perform simple commands. Autism-like behaviors, stereotypies, and bruxism were noted during the examination. WES revealed two undescribed variants of unknown clinical significance in the HTT gene, presumably associated with the patient's phenotype (c.2350C>T and c.8440C>A). Medical re-examination of parents revealed that the patient inherited these variants from her father and mother. Lopes−Maciel−Rodan syndrome was diagnosed based on overlapping clinical findings and the follow-up genetic examination of parents. Our finding expands the number of reported LOMARS cases and provides new insights into the genetic basis of the disease.

Molecular Genetics and Pathogenesis of the Floating Harbor Syndrome: Case Report of Long-Term Growth Hormone Treatment and a Literature Review.

Introduction: Floating Harbor syndrome (FHS) is an extremely rare disorder, with slightly more than a hundred cases reported worldwide. FHS is caused by heterozygous mutations in the SRCAP gene; however, little is known about the pathogenesis of FHS or the effectiveness of its treatment. Methods: Whole-exome sequencing (WES) was performed for the definitive molecular diagnosis of the disease. Identified variants were validated using Sanger sequencing. In addition, systematic literature and public data on genetic variation in SRCAP and the effects of growth hormone (GH) treatment was conducted. Results: We herein report the first case of FHS in the Russian Federation. The male proband presented with most of the typical phenotypic features of FHS, including short stature, skeletal and facial features, delayed growth and bone age, high pitched voice, and intellectual impairment. The proband also had partial growth hormone deficiency. We report the history of treatment of the proband with GH, which resulted in modest improvement in growth prior to puberty. WES revealed a pathogenic c.7466C>G (p.Ser2489*) mutation in the last exon of the FHS-linked SRCAP gene. A systematic literature review and analysis of available genetic variation datasets highlighted an unusual distribution of pathogenic variants in SRCAP and confirmed the lack of pathogenicity for variants outside of exons 33 and 34. Finally, we suggested a new model of FHS pathogenesis which provides possible basis for the dominant negative nature of FHS-causing mutations and explains limited effects of GH treatment in FHS. Conclusion: Our findings expand the number of reported FHS cases and provide new insights into disease genetics and the efficiency of GH therapy for FHS patients.

RNA Sequencing of Whole Blood Defines the Signature of High Intensity Exercise at Altitude in Elite Speed Skaters.

Although high altitude training has been increasingly popular among endurance athletes, the molecular and cellular bases of this adaptation remain poorly understood. We aimed to define the underlying physiological changes and screen for potential biomarkers of adaptation using transcriptional profiling of whole blood. Seven elite female speed skaters were profiled on the 18th day of high-altitude adaptation. Whole blood RNA-seq before and after an intense 1 h skating bout was used to measure gene expression changes associated with exercise. In order to identify the genes specifically regulated at high altitudes, we have leveraged the data from eight previously published microarray datasets studying blood expression changes after exercise at sea level. Using cell type-specific signatures, we were able to deconvolute changes of cell type abundance from individual gene expression changes. Among these were PHOSPHO1, with a known role in erythropoiesis, and MARC1 with a role in endogenic NO metabolism. We find that platelet and erythrocyte counts uniquely respond to altitude exercise, while changes in neutrophils represent a more generic marker of intense exercise. Publicly available data from both single cell atlases and exercise-related blood profiling dramatically increases the value of whole blood RNA-seq for the dynamic evaluation of physiological changes in an athlete's body.

Identification of Genetic Risk Factors of Severe COVID-19 Using Extensive Phenotypic Data: A Proof-of-Concept Study in a Cohort of Russian Patients.

The COVID-19 pandemic has drawn the attention of many researchers to the interaction between pathogen and host genomes. Over the last two years, numerous studies have been conducted to identify the genetic risk factors that predict COVID-19 severity and outcome. However, such an analysis might be complicated in cohorts of limited size and/or in case of limited breadth of genome coverage. In this work, we tried to circumvent these challenges by searching for candidate genes and genetic variants associated with a variety of quantitative and binary traits in a cohort of 840 COVID-19 patients from Russia. While we found no gene- or pathway-level associations with the disease severity and outcome, we discovered eleven independent candidate loci associated with quantitative traits in COVID-19 patients. Out of these, the most significant associations correspond to rs1651553 in MYH14p = 1.4 × 10-7), rs11243705 in SETX (p = 8.2 × 10-6), and rs16885 in ATXN1 (p = 1.3 × 10-5). One of the identified variants, rs33985936 in SCN11A, was successfully replicated in an independent study, and three of the variants were found to be associated with blood-related quantitative traits according to the UK Biobank data (rs33985936 in SCN11A, rs16885 in ATXN1, and rs4747194 in CDH23). Moreover, we show that a risk score based on these variants can predict the severity and outcome of hospitalization in our cohort of patients. Given these findings, we believe that our work may serve as proof-of-concept study demonstrating the utility of quantitative traits and extensive phenotyping for identification of genetic risk factors of severe COVID-19.

Basic Predictive Risk Factors for Cytokine Storms in COVID-19 Patients.

Objective: A critical role in coronavirus disease 2019 (COVID-19) pathogenesis is played by immune dysregulation that leads to a generalized uncontrolled multisystem inflammatory response, caused by overproduction of proinflammatory cytokines, known as "a cytokine storm" (CS), strongly associated with a severe course of disease. The aim of this study is to identify prognostic biomarkers for CS development in COVID-19 patients and integrate them into a prognostic score for CS-associated risk applicable to routine clinical practice. Materials and Methods: The authors performed a review of 458 medical records from COVID-19 patients (241 men and 217 women aged 60.0 ± 10.0) who received treatment in the St. Petersburg State Budgetary Institution of Healthcare City Hospital 40 (City Hospital 40, St. Petersburg), from Apr. 18, 2020 to Nov. 21, 2020. The patients were split in two groups: one group included 100 patients with moderate disease symptoms; the other group included 358 patients with progressive moderately severe, severe, and extremely severe disease. The National Early Warning Score (NEWS) score was used alongside with clinical assessment, chest computed tomographic (CT) scans, electrocardiography (ECG), and lab tests, like ferritin, C-reactive protein (CRP), interleukin (IL)-6, lactate dehydrogenase (LDH), and D-dimer. Results: The basic risk factors for cytokine storms in COVID-19 patients are male gender, age over 40 years, positive test result for replicative severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA, absolute lymphocyte count, dynamics in the NEWS score, as well as LDH, D-dimer, ferritin, and IL-6 levels. These clinical and instrumental findings can be also used as laboratory biomarkers for diagnosis and dynamic monitoring of cytokine storms. The suggested prognostic scale (including the NEWS score dynamics; serum IL-6 greater than 23 pg/ml; serum CRP 50 mg/L or greater; absolute lymphocyte count less than 0.72 × 109/L; positive test result for replicative coronavirus (SARS-CoV-2) RNA; age 40 years and over) is a useful tool to identify patients at a high risk for cytokine storm, requiring an early onset of anti-inflammatory therapy.

Identification of novel variants in the LDLR gene in Russian patients with familial hypercholesterolemia using targeted sequencing.

Familial hypercholesterolemia (FH) is caused by mutations in various genes, including the LDLR, APOB and PSCK9 genes; however, the spectrum of these mutations in Russian individuals has not been fully investigated. In the present study, mutation screening was performed on the LDLR gene and other FH-associated genes in patients with definite or possible FH, using next-generation sequencing. In total, 59 unrelated patients were recruited and sorted into two separate groups depending on their age: Adult (n=31; median age, 49; age range, 23-70) and children/adolescent (n=28; median age, 11; age range, 2-21). FH-associated variants were identified in 18 adults and 25 children, demonstrating mutation detection rates of 58 and 89% for the adult and children/adolescent groups, respectively. In the adult group, 13 patients had FH-associated mutations in the LDLR gene, including two novel variants [NM_000527.4: c.433_434dupG p.(Val145Glyfs*35) and c.1186G>C p.(Gly396Arg)], 3 patients had APOB mutations and two had ABCG5/G8 mutations. In the children/adolescent group, 21 patients had FH-causing mutations in the LDLR gene, including five novel variants [NM_000527.4: c.325T>G p.(Cys109Gly), c.401G>C p.(Cys134Ser), c.616A>C p.(Ser206Arg), c.1684_1691delTGGCCCAA p.(Pro563Hisfs*14) and c.940+1_c.940+4delGTGA], and 2 patients had APOB mutations, as well as ABCG8 and LIPA mutations, being found in different patients. The present study reported seven novel LDLR variants considered to be pathogenic or likely pathogenic. Among them, four missense variants were located in the coding regions, which corresponded to functional protein domains, and two frameshifts were identified that produced truncated proteins. These variants were observed only once in different patients, whereas a splicing variant in intron 6 (c.940+1_c.940+4delGTGA) was detected in four unrelated individuals. Previously reported variants in the LDLR, APOB, ABCG5/8 and LIPA genes were observed in 33 patients. The LDLR p.(Gly592Glu) variant was detected in 6 patients, representing 10% of the FH cases reported in the present study, thus it may be a major variant present in the Russian population. In conclusion, the present study identified seven novel variants of the LDLR gene and broadens the spectrum of mutations in FH-related genes in the Russian Federation.

Analysis of the Spectrum of ACE2 Variation Suggests a Possible Influence of Rare and Common Variants on Susceptibility to COVID-19 and Severity of Outcome.

OBJECTIVES: In March 2020, the World Health Organization declared that an infectious respiratory disease caused by a new severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2, causing coronavirus disease 2019 (COVID-19)] became a pandemic. In our study, we have analyzed a large publicly available dataset, the Genome Aggregation Database (gnomAD), as well as a cohort of 37 Russian patients with COVID-19 to assess the influence of different classes of genetic variants in the angiotensin-converting enzyme-2 (ACE2) gene on the susceptibility to COVID-19 and the severity of disease outcome. RESULTS: We demonstrate that the European populations slightly differ in alternative allele frequencies at the 2,754 variant sites in ACE2 identified in the gnomAD database. We find that the Southern European population has a lower frequency of missense variants and slightly higher frequency of regulatory variants. However, we found no statistical support for the significance of these differences. We also show that the Russian population is similar to other European populations when comparing the frequencies of the ACE2 variants. Evaluation of the effect of various classes of ACE2 variants on COVID-19 outcome in a cohort of Russian patients showed that common missense and regulatory variants do not explain the differences in disease severity. At the same time, we find several rare ACE2 variants (including rs146598386, rs73195521, rs755766792, and others) that are likely to affect the outcome of COVID-19. Our results demonstrate that the spectrum of genetic variants in ACE2 may partially explain the differences in severity of the COVID-19 outcome.

Related expression of TRKA and P75 receptors and the changing copy number of MYC-oncogenes determine the sensitivity of brain tumor cells to the treatment of the nerve growth factor in combination with cisplatin and temozolomide.

Objectives Oncological diseases are an urgent medical and social problem. The chemotherapy induces not only the death of the tumor cells but also contributes to the development of their multidrug resistance and death of the healthy cells and tissues. In this regard, the search for the new pharmacological substances with anticancer activity against drug-resistant tumors is of utmost importance. In the present study we primarily investigated the correlation between the expression of TrkA and p75 receptors with the nerve growth factor (NGF) and cisplatin or temozolomide sensitivity of anaplastic astrocytoma (AA), glioblastoma (GB) and medulloblastoma (MB) cell cultures. We then evaluated the changing of copy numbers of MYCC and MYCN and its correlation with cytotoxicity index (CI) in MB cells under NGF exposition. Methods The primary cell cultures were obtained from the tumor biopsy samples of the patients with AA (n=5), GB (n=7) or MB (n=25) prior to radiotherapy and chemotherapy. The cytotoxicity effect of NGF and its combinations with cisplatin or temozolomide, the relative expression of TrkA and p75 receptors, its correlations with CI in AA, GB and MB primary cell cultures were studied by trypan blue cytotoxicity assay and immunofluorescence staining respectively. The effect of NGF on MYCC and MYCN copy numbers in MB cell cultures was studied by fluorescence in situ hybridization. Results We found that the expression of TrkA and p75 receptors (p=0.03) and its ratio (p=0.0004) depends on the sensitivity of AA and GB cells to treatment with NGF and its combinations with cisplatin or temozolomide. NGF reduces (p<0.05) the quantity of MB cells with six or eight copies of MYCN and three or eight copies of MYCC. Besides, NGF increases (p<0.05) the quantity of MB cells containing two copies of both oncogenes. The negative correlation (r=-0.65, p<0.0001) is established between MYCC average copy numbers and CI of NGF in MB cells. Conclusions The relative expression of NGF receptors (TrkA/p75) and its correlation with CI of NGF and its combinations in AA and GB cells point to the mechanism involving a cell death signaling pathway. NGF downregulates (p<0.05) some increased copy numbers of MYCC and MYCN in the human MB cell cultures, and upregulates normal two copies of both oncogenes (p<0.05).

Systematic dissection of biases in whole-exome and whole-genome sequencing reveals major determinants of coding sequence coverage.

Advantages and diagnostic effectiveness of the two most widely used resequencing approaches, whole exome (WES) and whole genome (WGS) sequencing, are often debated. WES dominated large-scale resequencing projects because of lower cost and easier data storage and processing. Rapid development of 3rd generation sequencing methods and novel exome sequencing kits predicate the need for a robust statistical framework allowing informative and easy performance comparison of the emerging methods. In our study we developed a set of statistical tools to systematically assess coverage of coding regions provided by several modern WES platforms, as well as PCR-free WGS. We identified a substantial problem in most previously published comparisons which did not account for mappability limitations of short reads. Using regression analysis and simple machine learning, as well as several novel metrics of coverage evenness, we analyzed the contribution from the major determinants of CDS coverage. Contrary to a common view, most of the observed bias in modern WES stems from mappability limitations of short reads and exome probe design rather than sequence composition. We also identified the ~ 500 kb region of human exome that could not be effectively characterized using short read technology and should receive special attention during variant analysis. Using our novel metrics of sequencing coverage, we identified main determinants of WES and WGS performance. Overall, our study points out avenues for improvement of enrichment-based methods and development of novel approaches that would maximize variant discovery at optimal cost.

Related expression of TRKA and P75 receptors and the changing copy number of MYC-oncogenes determine the sensitivity of brain tumor cells to the treatment of the nerve growth factor in combination with cisplatin and temozolomide.

OBJECTIVES: Oncological diseases are an urgent medical and social problem. The chemotherapy induces not only the death of the tumor cells but also contributes to the development of their multidrug resistance and death of the healthy cells and tissues. In this regard, the search for the new pharmacological substances with anticancer activity against drug-resistant tumors is of utmost importance. In the present study we primarily investigated the correlation between the expression of TrkA and p75 receptors with the nerve growth factor (NGF) and cisplatin or temozolomide sensitivity of anaplastic astrocytoma (AA), glioblastoma (GB) and medulloblastoma (MB) cell cultures. We then evaluated the changing of copy numbers of MYCC and MYCN and its correlation with cytotoxicity index (CI) in MB cells under NGF exposition. METHODS: The primary cell cultures were obtained from the tumor biopsy samples of the patients with AA (n=5), GB (n=7) or MB (n=25) prior to radiotherapy and chemotherapy. The cytotoxicity effect of NGF and its combinations with cisplatin or temozolomide, the relative expression of TrkA and p75 receptors, its correlations with CI in AA, GB and MB primary cell cultures were studied by trypan blue cytotoxicity assay and immunofluorescence staining respectively. The effect of NGF on MYCC and MYCN copy numbers in MB cell cultures was studied by fluorescence in situ hybridization. RESULTS: We found that the expression of TrkA and p75 receptors (p=0.03) and its ratio (p=0.0004) depends on the sensitivity of AA and GB cells to treatment with NGF and its combinations with cisplatin or temozolomide. NGF reduces (p<0.05) the quantity of MB cells with six or eight copies of MYCN and three or eight copies of MYCC. Besides, NGF increases (p<0.05) the quantity of MB cells containing two copies of both oncogenes. The negative correlation (r=-0.65, p<0.0001) is established between MYCC average copy numbers and CI of NGF in MB cells. CONCLUSIONS: The relative expression of NGF receptors (TrkA/p75) and its correlation with CI of NGF and its combinations in AA and GB cells point to the mechanism involving a cell death signaling pathway. NGF downregulates (p<0.05) some increased copy numbers of MYCC and MYCN in the human MB cell cultures, and upregulates normal two copies of both oncogenes (p<0.05).

The spectrum of pathogenic variants of the ATP7B gene in Wilson disease in the Russian Federation.

BACKGROUND: Wilson's disease (WD) is a rare inherited disorder caused by mutations in the ATP7B gene resulting in copper accumulation in different organs. However, data on ATP7B mutation spectrum in Russia and worldwide are insufficient and contradictory. The objective of the present study was estimation of the frequency of ATP7B gene mutations in the Russian population of WD patients. MATERIALS AND METHODS: 75 WDpatients were examined by next-generation sequencing (NGS). A targeted panel NimbleGen SeqCap EZ Choice: 151012_HG38_CysFib_EZ_HX3 (ROCHE)was designed for analysis of ATP7B gene and possible modifier genes. Retrospective assessment of a diagnostic WD score (Leipzig, 2001) was also performed. RESULTS: 31 mutations in ATP7B gene were detected. Two most frequent mutations were c.3207C > A (51,85% of alleles) and c.3190 G > A (8,64% of alleles). Single rare mutations were detected in 29% of cases. In 96% cases mutations of both copies of the ATP7B were revealed. We also observed 3 novel potentially pathogenic variants which were not previously described (c.1870-8A > G, c.3655A > T (p.Ile1219Phe), c.3036dupC (p.Lys1013fs). For 25% of patients at the time of the manifestation the diagnosis WD could not be established using the earlier proposed diagnostic score. There was a remarkable delay in diagnosis for the majority of patients. Only 33% of patients WD was diagnosed in three months after the first symptoms, 29%patients - in 3-12 months, 30% - in 1-10 years, in 8% - more than 10 years. Generally, clinical appearance of WD may be rather variable at manifestation and genetic profiling at this step is the only way to confirm the presence of WD.