Генетическая компонента многофакторных заболеваний интенсивно изучается на протяжении многих лет, но до сих пор в этой области остается очень много вопросов. В последние годы появились исследования в области “менделевского кода” – гипотезы, постулирующей существование связи между менделевскими (моногенными) и многофакторными патологиями: полиморфные варианты в генах менделевских заболеваний могут оказаться значимыми для многофакторных патологий, в которых задействованы те же биохимические пути. В настоящем обзоре через призму гипотезы “менделевского кода” представлен взгляд на группу генов, которые могут оказаться перспективными генами-кандидатами широкого спектра патологических состояний, а именно, генов белков систем репарации ДНК. В качестве примера рассмотрены белки BRСA1-ассоциированного комплекса наблюдения за геномом (BASC). Обсуждаются основные функции белков этого комплекса в процессах репарации двухцепочечных разрывов ДНК (ATM, MRE11, NBN, RAD50, BRCA1, BLM) и мисматч-репарации (MSH2, MSH6, MLH1, PMS2, RF-C, PCNA); вовлеченность этих белков в функционирование митохондрий; участие белков BASC в развитии адаптивного иммунного ответа...

Patients with cystic fibrosis (CF) need costly medical care and adequate therapy with expensive medicinal products. Tigerase® is the first biosimilar of dornase alfa, developed by the lead Russian biotechnology company GENERIUM. The aim of the manuscript to present post hoc sub-analysis of patients' data with cystic fibrosis and severe pulmonary impairment of a larger comparative study (phase III open label, prospective, multi-centre, randomized study (NCT04468100)) of a generic version of recombinant human DNase Tigerase to the only comparable drug, Pulmozym.

Understanding the mechanisms underlying the connection between somatic mosaicism and cardiovascular disease is likely essential for the future of personalized medicine. This article is aimed at providing data on somatic mosaicism in human carotid atherosclerosis. An advanced carotid atherosclerotic plaque and white blood cells were collected simultaneously from each patient (eight Slavic males, aged 67 ± 3.8 years [mean ± SD]) to assess the spectrum of germline and somatic genetic variants. Exome sequencing of DNA from the samples was performed with the SureSelect Clinical Research Exome Enrichment Kit (Agilent Technologies) and HiSeq 1500 (Illumina). The dataset contains germline and somatic single-nucleotide variants and small indels identified in the advanced carotid atherosclerotic plaque and white blood cells of each patient. This dataset does not include copy number variants owing to a lack of suitable tools for reliable calculation of copy numbers from exome sequencing data on cancer-unrelated samples. The dataset should help to understand somatic mosaicism in cardiovascular diseases and to identify copy number variants by means of more appropriate newer tools in the future.

Wilson's disease is a rare autosomal recessive disorder of copper metabolism. The copper accumulation in the viscera appears due to the functional impairment of copper-transporting ATPase, which is encoded by the ATP7B gene. In this study, PBMCs of a patient with two ATP7B mutations were reprogrammed. The first mutation is a missense mutation p.H1069Q, which is the most frequent mutation in the human population. At the same time, the second one is a frameshift mutation p.Lys1013fs. The generated iPSC line had a normal karyotype, maintained the original genotype, expressed pluripotency markers, and demonstrated the ability to differentiate into derivatives of the three germ layers.

Comparative analysis of multilocus imprinting disturbances (MLIDs) in miscarriages from women with sporadic (SPL) and recurrent pregnancy loss (RPL) and identification of variants in the imprinting control gene NLRP7 that may lead to MLIDs.

Alzheimer's disease (AD) is a neurodegenerative disorder, and represents the most common cause of dementia. In this study, we performed several different analyses to detect loci involved in development of the late onset AD in the Russian population. DNA samples from 472 unrelated subjects were genotyped for 63 SNPs using iPLEX Assay and real-time PCR. We identified five genetic loci that were significantly associated with LOAD risk for the Russian population (TOMM40 rs2075650, APOE rs429358 and rs769449, NECTIN rs6857, APOE ε4). The results of the analysis based on comparison of the haplotype frequencies showed two risk haplotypes and one protective haplotype. The GMDR analysis demonstrated three significant models as a result: a one-factor, a two-factor and a three-factor model. A protein-protein interaction network with three subnetworks was formed for the 24 proteins. Eight proteins with a large number of interactions are identified: APOE, SORL1, APOC1, CD33, CLU, TOMM40, CNTNAP2 and CACNA1C. The present study confirms the importance of the APOE-TOMM40 locus as the main risk locus of development and progress of LOAD in the Russian population. Association analysis and bioinformatics approaches detected interactions both at the association level of single SNPs and at the level of genes and proteins.

Split reporter protein-based genetic section systems are widely used to identify and characterize protein-protein interactions (PPI). The assembly of split markers that antagonize toxins, rather than required for synthesis of missing metabolites, facilitates the seeding of high density of cells and selective growth. Here we present a newly developed split chloramphenicol acetyltransferase (split-CAT) -based genetic selection system. The N terminus fragment of CAT is fused downstream of the protein of interest and the C terminus fragment is tethered upstream to its postulated partner. We demonstrate the system's advantages for the study of PPIs. Moreover, we show that co-expression of a functional ubiquitylation cascade where the target and ubiquitin are tethered to the split-CAT fragments results in ubiquitylation-dependent selective growth. Since proteins do not have to be purified from the bacteria and due to the high sensitivity of the split-CAT reporter, detection of challenging protein cascades and post-translation modifications is enabled. In addition, we demonstrate that the split-CAT system responds to small molecule inhibitors and molecular glues (GLUTACs). The absence of ubiquitylation-dependent degradation and deubiquitylation in E. coli significantly simplify the interpretation of the results. We harnessed the developed system to demonstrate that like NEDD4, UBE3B also undergoes self-ubiquitylation-dependent inactivation. We show that self-ubiquitylation of UBE3B on K665 induces oligomerization and inactivation in yeast and mammalian cells respectively. Finally, we showcase the advantages of split-CAT in the study of human diseases by demonstrating that mutations in UBE3B that cause Kaufman oculocerebrofacial syndrome exhibit clear E. coli growth phenotypes.

Primary autosomal recessive microcephaly and Seckel syndrome spectrum disorders (MCPH-SCKS) include a heterogeneous group of autosomal recessive inherited diseases characterized by primary (congenital) microcephaly, the absence of visceral abnormalities, and a variable degree of cognitive impairment, short stature and facial dysmorphism. Recently, biallelic variants in the nuclear pore complex (NPC) component nucleoporin 85 gene (NUP85) were reported to cause steroid-resistant nephrotic syndrome (SRNS). Here, we report biallelic variants in NUP85 in two pedigrees with an MCPH-SCKS phenotype spectrum without SRNS, thereby expanding the phenotypic spectrum of NUP85-linked diseases. Structural analysis predicts the identified NUP85 variants cause conformational changes that could have an effect on NPC architecture or on its interaction with other NUPs. We show that mutant NUP85 is, however, associated with a reduced number of NPCs but unaltered nucleocytoplasmic compartmentalization, abnormal mitotic spindle morphology, and decreased cell viability and proliferation in one patient's cells. Our results also indicate the link of common cellular mechanisms involved in MCPH-SCKS spectrum disorders and NUP85-associated diseases. In addition to the previous studies, our results broaden the phenotypic spectrum of NUP85-linked human disease and propose a role for NUP85 in nervous system development.

Metabolic syndrome (MetS) is characterized by abdominal obesity, hyperglycaemia, dyslipidaemia and hypertension. FTO gene has been implicated in the pathogenesis of obesity, but the available scientific data concerning their relationship to antipsychotic drug-induced obesity and metabolic syndrome is still incomplete and inconsistent, which indicates that continuing the investigation of this gene's role is necessary.

Recent studies have focused on the potential role of epicardial adipose tissue (EAT) in the development of coronary artery disease (CAD). ABCA1 and ABCG1 transporters regulate cell cholesterol content and reverse cholesterol transport. We aimed to determine whether DNA methylation and mRNA levels of the ABCA1 and ABCG1 genes in EAT and subcutaneous adipose tissue (SAT) were associated with CAD.

Chromosomal mosaicism is a hallmark of early human embryo development. The last decade yielded an enormous amount of information about diversity and prevalence of mosaicism in preimplantation embryos due to progress in preimplantation genetic testing of aneuploidies (PGT-A) based exclusively on molecular karyotyping of trophectoderm biopsy. However, the inner cell mass karyotype is still missing for mosaic embryos affecting the success rate of assisted reproductive medicine. Here, a classification model of chromosomal mosaicism is proposed based on the analysis of the primary zygote karyotype, the timing and types of primary and secondary chromosome segregation errors, and the distribution of mosaic cell clones between different embryonic and extraembryonic compartments of the blastocyst. Five basic principles for mosaicism analysis are introduced, namely, the estimation of the primary zygote karyotype, the investigation of additional sample point, the requirement of the second time point analysis, the delineating of reciprocity of chromosome segregation, and comprehensive chromosome screening at the single-cell level. The suggested model allows the prediction of the inner cell mass karyotype of the blastocyst and its developmental potential based on information from trophectoderm biopsy and non-invasive PGT-A using blastocoele fluid sample or spent culture medium as additional sample and time points for analysis and considering the reciprocity as a basic process in chromosome segregation errors between daughter cells in postzygotic cell divisions.

Interpreting the clinical significance of small supernumerary marker chromosomes (sSMCs) in prenatal diagnosis is still an urgent problem in genetic counselling regarding the fate of a pregnancy. We present a case of prenatal diagnosis of mosaic sSMC(10) in a foetus with a normal phenotype. Comprehensive cytogenomic analyses by array-based comparative genomic hybridization (aCGH), sSMC microdissection with next-generation sequencing (NGS) of microdissected library, fluorescence in situ hybridization (FISH) with locus-specific and telomere-specific DNA probes and quantitative real-time PCR revealed that sSMC(10) had a ring structure and was derived from the pericentromeric region of chromosome 10 with involvement of the 10p11.21-p11.1 and 10q11.21-q11.23 at 1.243 Mb and 7.173 Mb in size, respectively. We observed a difference in the length of sSMC(10) between NGS data of the DNA library derived from a single copy of sSMC(10), and aCGH results that may indicate instability and structural mosaicism for ring chromosomes in foetal cells. The presence of a 9 Mb euchromatin region in the analysed sSMC(10) did not lead to clinical manifestations, and a healthy girl was born at term. We suggest that the ring structure of sSMCs could influence sSMC manifestations and should be taken into account in genetic counselling during prenatal diagnosis.

The methylation index of the LINE-1 promoter is one of the most commonly used markers for assessing the global level of genome methylation in various human cells and tissues. We developed an NGS-based protocol for DNA methylation analysis of the LINE-1 retrotransposon promoter. This approach allows assessment of the DNA methylation index of 19 CpG sites in the LINE-1 promoter that have the highest tissue- or tumor-specific variability. The method provides a DNA methylation profile for analyzing either the methylation index of each CpG site independently or the mean DNA methylation index across the LINE-1 promoter. The results obtained using the developed method corresponded well to the level of methylation assessed using a commercially available kit for DNA pyrosequencing. In addition, our method provides much more information: 1) the DNA methylation profile of a significant part of the LINE-1 promoter and 2) the level of DNA methylation at individual LINE-1 loci in the genome. The method of targeted bisulfite massive parallel sequencing of the human LINE-1 retrotransposon promoter can be used in large-scale studies of the global level of genome methylation in normal human cells or tumors. To accomplish this, we modified the targeted massive parallel sequencing method based on 16S Metagenomic Sequencing Library Preparation protocol (Illumina, USA) by: Introduction of the stage of bisulfite conversion of DNA. Development of specific primers for the LINE-1 sequence.

Quantification of DNA methylation in neoplastic cells is crucial both from mechanistic and diagnostic perspectives. However, such measurements are prone to different experimental biases. Polymerase chain reaction (PCR) bias results in an unequal recovery of methylated and unmethylated alleles at the sample preparation step. Post-PCR biases get introduced additionally by the readout processes. Correcting the biases is more practicable than optimising experimental conditions, as demonstrated previously. However, utilisation of our earlier developed algorithm strongly necessitates automation. Here, we present two R packages: rBiasCorrection, the core algorithms to correct biases; and BiasCorrector, its web-based graphical user interface frontend. The software detects and analyses experimental biases in calibration DNA samples at a single base resolution by using cubic polynomial and hyperbolic regression. The correction coefficients from the best regression type are employed to compensate for the bias. Three common technologies-bisulphite pyrosequencing, next-generation sequencing and oligonucleotide microarrays-were used to comprehensively test BiasCorrector. We demonstrate the accuracy of BiasCorrector's performance and reveal technology-specific PCR- and post-PCR biases. BiasCorrector effectively eliminates biases regardless of their nature, locus, the number of interrogated methylation sites and the detection method, thus representing a user-friendly tool for producing accurate epigenetic results.

Dysregulation of the oxidant-antioxidant system contributes to the pathogenesis of cerebral stroke (CS). Epigenetic changes of redox homeostasis genes, such as glutamate-cysteine ligase (GCLM), glutathione-S-transferase-P1 (GSTP1), thioredoxin reductase 1 (TXNRD1), and myeloperoxidase (MPO), may be biomarkers of CS. In this study, we assessed the association of DNA methylation levels of these genes with CS and clinical features of CS. We quantitatively analyzed DNA methylation patterns in the promoter or regulatory regions of 4 genes (GCLM, GSTP1, TXNRD1, and MPO) in peripheral blood leukocytes of 59 patients with CS in the acute phase and in 83 relatively healthy individuals (controls) without cardiovascular and cerebrovascular diseases. We found that in both groups, the methylation level of CpG sites in genes TXNRD1 and GSTP1 was ≤ 5%. Lower methylation levels were registered at a CpG site (chr1:94,374,293, GRCh37 [hg19]) in GCLM in patients with ischemic stroke compared with the control group (9% [7%; 11.6%] (median and interquartile range) versus 14.7% [10.4%; 23%], respectively, p < 0.05). In the leukocytes of patients with CS, the methylation level of CpG sites in the analyzed region of MPO (chr17:56,356,470, GRCh3 [hg19]) on average was significantly lower (23.5% [19.3%; 26.7%]) than that in the control group (35.6% [30.4%; 42.6%], p < 0.05). We also found increased methylation of MPO in smokers with CS (27.2% [23.5%; 31.1%]) compared with nonsmokers with CS (21.7% [18.1%; 24.8%]). Thus, hypomethylation of CpG sites in GCLM and MPO in blood leukocytes is associated with CS in the acute phase.