The SLC26A4 gene is one of the key genes involved in the etiology of hearing loss. It encodes pendrin, a transmembrane transporter protein functioning as a multifunctional anion exchanger. About 600 pathogenic SLC26A4 variants are known to cause either nonsyndromic recessive hearing loss (DFNB4) or Pendred syndrome (hearing loss and thyroid dysfunction). While most pathogenic variants occur in coding regions and disrupt pendrin structure or function, about 25% are thought to impair splicing. For many, pathogenicity has been assessed only in silico, with limited experimental confirmation. We identified several novel and rare SLC26A4 variants in patients with hearing loss from the Tyva and Altai Republics (Southern Siberia, Russia). Based on splicing predictions, six variants—intronic c.2034+1G>A, c.1545-168A>G, c.1708-125T>C, c.1708-18T>A, c.1804-31C>T, and exonic c.942A>G—were selected for analysis using a minigene assay. The results of in vitro analysis only partially matched in silico predictions: c.2034+1G>A caused aberrant splicing; c.1708-18T>A led to exon 16 skipping only in a small proportion of transcripts; the remaining variants showed no detectable splicing effect. These findings underscore the need for integrating in silico predictions with in vitro validation to accurately assess the functional impact of genetic variants, enabling their correct interpretation and reliable molecular diagnosis.

Targeting DNA repair pathways is an important strategy in anticancer therapy. However, the unrevealed interactions between different DNA repair systems may interfere with the desired therapeutic effect. Among DNA repair systems, BER and NHEJ protect genome integrity through the entire cell cycle. BER is involved in the repair of DNA base lesions and DNA single-strand breaks (SSBs), while NHEJ is responsible for the repair of DNA double-strand breaks (DSBs). Previously, we showed that BER deficiency leads to downregulation of NHEJ gene expression. Here, we studied BER’s response to NHEJ deficiency induced by knockdown of NHEJ scaffold protein XRCC4 and compared the knockdown effects in normal (TIG-1) and hTERT-modified cells (NBE1). We investigated the expression of the XRCC1, LIG3, and APE1 genes of BER and LIG4; the Ku70/Ku80 genes of NHEJ at the mRNA and protein levels; as well as p53, Sp1 and PARP1. We found that, in both cell lines, XRCC4 knockdown leads to a decrease in the mRNA levels of both BER and NHEJ genes, though the effect on protein level is not uniform. XRCC4 knockdown caused an increase in p53 and Sp1 proteins, but caused G1/S delay only in normal cells. Despite the increased p53 protein, p21 did not significantly increase in NBE1 cells with overexpressed hTERT, and this correlated with the absence of G1/S delay in these cells. The data highlight the regulatory function of the XRCC4 scaffold protein and imply its connection to a transcriptional regulatory network or mRNA metabolism.

Pallister-Killian syndrome (PKS) is a rare inherited disease with multiple congenital anomalies, profound intellectual disability, and the presence in the karyotype of sSMC - i(12)(p10). The frequency of PKS may be underestimated due to problems with cytogenetic diagnosis caused by tissue-specific mosaicism and usually a low percentage of peripheral blood cells containing sSMC. Such tissue-specific mosaicism also complicates a detailed analysis of the sSMC, which, along with the assessment of mosaicism in different tissues, is an important part of cytogenetic diagnosis in PKS. Unfortunately, a full-fledged diagnosis in PKS is either practically impossible or complicated. On the one hand, this is due to problems with the biopsy of various tissues (skin biopsy with fibroblast culture is most often used in practice); on the other - a low percentage of dividing peripheral blood cells containing sSMC, which often significantly complicates the analysis of its composition and organization. In the present study, a detailed analysis of sSMC was carried out in a patient with a characteristic clinical picture of PKS. A relatively high percentage of peripheral blood cells with sSMC (50%) made it possible to perform a detailed molecular cytogenetic analysis of de novo sSMC using chromosomal in situ suppression hybridization (CISS-hybridization), multicolor FISH (mFISH), multicolor chromosome banding (MCB), array CGH (aCGH), and quantitative real-time PCR (qPCR), and short tandem repeat (STR) - analysis. As a result, it was found that the sSMC is not a typical PKS derivative of chromosome 12. In contrast to the classical i(12)(p10) for PKS, the patient’s cells contained an acrocentric chromosome consisting of 12p material. Clusters of telomeric repeats were found at the both ends of the sSMC. Furthemore, the results of aCGH and qPCR indicate the presence of interstitial 8.9 Mb duplication at 12p13.1-p12.1 within the sSMC, which leads to different representations of DNA from different segments of 12p within cells containing sSMC. The obtained data raise the question of the instability of the sSMC and, as a consequence, the possible presence of additional rearrangements, which, in traditional cytogenetic analysis of patients with PKS, are usually described as i(12)(p10).

Pathogenic variants in the SLC26A4 gene leading to nonsyndromic recessive deafness (DFNB4), or Pendred syndrome, are some of the most common causes of hearing loss worldwide. Earlier, we found a high proportion of SLC26A4-related hearing loss with prevailing pathogenic variant c.919-2A>G (69.3% among all mutated SLC26A4 alleles that have been identified) in Tuvinian patients belonging to the indigenous Turkic-speaking Siberian people living in the Tyva Republic (Southern Siberia, Russia), which implies a founder effect in the accumulation of c.919-2A>G in Tuvinians. To evaluate a possible common origin of c.919-2A>G, we genotyped polymorphic STR and SNP markers, intragenic and flanking SLC26A4, in patients homozygous for c.919-2A>G and in healthy controls. The common STR and SNP haplotypes carrying c.919-2A>G were revealed, which convincingly indicates the origin of c.919-2A>G from a single ancestor, supporting a crucial role of the founder effect in the c.919-2A>G prevalence in Tuvinians. Comparison analysis with previously published data revealed the identity of the small SNP haplotype (~4.5 kb) in Tuvinian and Han Chinese carriers of c.919-2A>G, which suggests their common origin from founder chromosomes. We assume that c.919-2A>G could have originated in the geographically close territories of China or Tuva and subsequently spread to other regions of Asia. In addition, the time intervals of the c.919-2A>G occurrence in Tuvinians were roughly estimated.

One of the most common forms of genetic deafness has been predominantly associated with pathogenic variants in the GJB2 gene, encoding transmembrane protein connexin 26 (Cx26). The Cx26 molecule consists of an N-terminal domain (NT), four transmembrane domains (TM1-TM4), two extracellular loops (EL1 and EL2), a cytoplasmic loop, and a C-terminus (CT). Pathogenic variants in the GJB2 gene, resulting in amino acid substitutions scattered across the Cx26 domains, lead to a variety of clinical outcomes, including the most common non-syndromic autosomal recessive deafness (DFNB1A), autosomal dominant deafness (DFNA3A), as well as syndromic forms combining hearing loss and skin disorders. However, for rare and poorly documented variants, information on the mode of inheritance is often lacking. Numerous in vitro studies have been conducted to elucidate the functional consequences of pathogenic GJB2 variants leading to amino acid substitutions in different domains of Cx26 protein. In this work, we summarized all available data on a mode of inheritance of pathogenic GJB2 variants leading to amino acid substitutions and reviewed published information on their functional effects, with an emphasis on their localization in certain Cx26 domains.

The proliferative activity of populations of stromal cells (fibroblasts) obtained from human corneal lenticles under conditions of their differentiation into keratocytes was studied. It was shown that during differentiation, the number of dividing fibroblasts and the frequency of divisions, and motor activity of these cells (speed of movement along the cell trajectory and the length of the trajectory) sharply decreased. These findings indicate a decrease in the proliferative activity of fibroblasts under conditions of their differentiation and transformation into keratocytes. A period of 17 days is sufficient for differentiation of corneal fibroblasts into keratocytes.

Detection and precise genomic mapping of balanced chromosomal abnormalities in patients with impaired fertility or a clinical phenotype represent a challenge for current cytogenomics owing to difficulties with precise breakpoint localization in the regions enriched for DNA repeats and high genomic variation in such regions. Here, we present a comprehensive cytogenomic approach to breakpoint mapping in a rare paracentric inversion on 10q (in a patient with oligoasthenoteratozoospermia and necrozoospermia) that does not affect other phenotype traits. Multicolor banding, chromosomal microarray analysis, chromosome microdissection with reverse painting, and single-copy sequencing of the rearranged chromosome were performed to determine the length and position of the inverted region as well as to rule out a genetic imbalance at the breakpoints. As a result, a paracentric 19.251 Mbp inversion at 10q22.2q23.3 was described. The most probable location of the breakpoints was predicted using the hg38 assembly. The problems of genetic counseling associated with enrichment for repeats and high DNA variability of usual breakpoint regions were discussed. Possible approaches for cytogenomic assessment of couples with balanced chromosome rearrangements and problems like reproductive failures were considered and suggested as useful part of effective genetic counseling.

The generally accepted theory of the genetic drift of mitochondrial alleles during mammalian ontogenesis is based on the presence of a selective bottleneck in the female germline. However, there is a variety of different theories on the pathways of genetic regulation of mitochondrial DNA (mtDNA) dynamics in oogenesis and adult somatic cells. The current review summarizes present knowledge on the natural mechanisms of mitochondrial genome elimination during mammalian development. We also discuss the variety of existing and developing methodologies for artificial manipulation of the mtDNA heteroplasmy level. Understanding of the basics of mtDNA dynamics will shed the light on the pathogenesis and potential therapies of human diseases associated with mitochondrial dysfunction.

Selective regulation of gene expression by means of RNA interference has revolutionized molecular biology. This approach is not only used in fundamental studies on the roles of particular genes in the functioning of various organisms, but also possesses practical applications. A variety of methods are being developed based on gene silencing using dsRNA-for protecting agricultural plants from various pathogens, controlling insect reproduction, and therapeutic techniques related to the oncological disease treatment. One of the main problems in this research area is the successful delivery of exogenous dsRNA into cells, as this can be greatly affected by the localization or origin of tumor. This overview is dedicated to describing the latest advances in the development of various transport agents for the delivery of dsRNA fragments for gene silencing, with an emphasis on cancer treatment.

Mutations in the GJB2 gene encoding transmembrane protein connexin 26 (Cx26) are the most common cause for hearing loss worldwide. Cx26 plays a crucial role in the ionic and metabolic homeostasis in the inner ear, indispensable for normal hearing process. Different pathogenic mutations in the GJB2 gene can affect all stages of the Cx26 life cycle and result in nonsyndromic autosomal recessive (DFNB1) or dominant (DFNA3) deafness and syndromes associating hearing loss with skin disorders. This study aims to elucidate the functional consequences of a rare GJB2 variant c.516G>C (p.Trp172Cys) found with high frequency in deaf patients from indigenous populations of Southern Siberia (Russia). The substitution c.516G>C leads to the replacement of tryptophan at a conserved amino acid position 172 with cysteine (p.Trp172Cys) in the second extracellular loop of Cx26 protein. We analyzed the subcellular localization of mutant Cx26-p.Trp172Cys protein by immunocytochemistry and the hemichannels permeability by dye loading assay. The GJB2 knockout HeLa cell line has been generated using CRISPR/Cas9 genome editing tool. Subsequently, the HeLa transgenic cell lines stably expressing different GJB2 variants (wild type and mutations associated with hearing loss) were established based on knockout cells and used for comparative functional analysis. The impaired trafficking of mutant Cx26-p.Trp172Cys protein to the plasma membrane and reduced hemichannels permeability support the pathogenic effect of the c.516G>C (p.Trp172Cys) variant and its association with nonsyndromic hearing loss. Our data contribute to a better understanding of the role of mutations in the second extracellular loop of Cx26 protein in pathogenesis of deafness.

The gene composition, function and evolution of B-chromosomes (Bs) have been actively discussed in recent years. However, the additional genomic elements are still enigmatic. One of Bs mysteries is their spatial organization in the interphase nucleus. It is known that heterochromatic compartments are not randomly localized in a nucleus. The purpose of this work was to study the organization and three-dimensional spatial arrangement of Bs in the interphase nucleus. Using microdissection of Bs and autosome centromeric heterochromatic regions of the yellow-necked mouse (Apodemus flavicollis) we obtained DNA probes for further two-dimensional (2D)- and three-dimensional (3D)- fluorescence in situ hybridization (FISH) studies. Simultaneous in situ hybridization of obtained here B-specific DNA probes and autosomal C-positive pericentromeric region-specific probes further corroborated the previously stated hypothesis about the pseudoautosomal origin of the additional chromosomes of this species. Analysis of the spatial organization of the Bs demonstrated the peripheral location of B-specific chromatin within the interphase nucleus and feasible contact with the nuclear envelope (similarly to pericentromeric regions of autosomes and sex chromosomes). It is assumed that such interaction is essential for the regulation of nuclear architecture. It also points out that Bs may follow the same mechanism as sex chromosomes to avoid a meiotic checkpoint.

The nucleotide excision repair (NER) is one of the main repair systems present in the cells of living organisms. It is responsible for the removal of a wide range of bulky DNA lesions. We succeeded in developing a method for assessing the efficiency of NER in the cell (ex vivo), which is a method based on the recovery of TagRFP fluorescent protein production through repair of the damage that blocks the expression of the appropriate gene. Our constructed plasmids containing bulky nFlu or nAnt lesions near the tagrfp gene promoter were shown to undergo repair in eukaryotic cells (HEK 293T) and that they can be used to analyze the efficiency of NER ex vivo. A comparative analysis of the time dependence of fluorescent cells accumulation after transfection with nFlu- and nAnt-DNA revealed that there are differences in how efficient their repair by the NER system of HEK 293T cells can be. The method can be used to assess the cell repair status and the repair efficiency of different structural damages.

В работе приводятся результаты комплексной оценки патогенетической значимости нового варианта с.516G>C (p.Trp172Cys) гена GJB2, выявленного с высокой частотой у больных с потерей слуха из Республик Тыва и Алтай (Южная Сибирь). Совокупность полученных данных свидетельствует в пользу ассоциации этого варианта с наследственной глухотой вследствие его повреждающего эффекта на структуру и функционирование белка коннексина 26 (Cx26). We present the results of a complex assessment of the pathogenetic significance of novel variant c.516G>C (p.Trp172Cys) of the GJB2 gene found with high frequency in deaf patients from the Tyva and Altai Republics. Several lines of evidences support pathogenicity of this variant due to its damaging effect on the structure and functioning of connexin 26 (Cx26).

The HIF-2 a subunit is involved in regulation of transcription factors, controlling the self-renewal of human pluripotent stem cells, embryonic development of the cardiovascular system and the regulation of angiogenesis by transcriptional activation of angiogenic cascades in physiological and pathological processes. Currently, modulation of HIF-2a expression is considered as a promising strategy for the treatment of ischemic and cancer diseases. However, the problem of choosing the optimal methods of effective regulation of HIF-2a remains. The aim of this study is to obtain human embryonic stem cells with increased expression of HIF-2a at normal oxygen concentration due to silencing of INT6, the regulator of HIF-2a. In this study, we obtained genetically modified human embryonic stem cells with increased expression of HIF-2a under atmospheric oxygen conditions. The approach used is based on a CRISPR/Cas9-mediated deletion of a part of the INT6 gene, an HIF-2a inhibitor. A study of the resulting genetically modified human embryonic stem cells will contribute to an understanding of the connection between hypoxia and pluripotency. Obtaining endothelial derivatives of pluripotent stem cells with increased expression of HIF-2a and enhanced regenerative potential may become the basis for the development of promising strategies for treatment of ischemic diseases.

It has long been known that defects in the structure of the mitochondrial genome can cause various neuromuscular and neurodegenerative diseases. Nevertheless, at present there is no effective method for treating mitochondrial diseases. The major problem with the treatment of such diseases is associated with mitochondrial DNA (mtDNA) heteroplasmy. It means that due to a high copy number of the mitochondrial genome, mutant copies of mtDNA coexist with wild-type molecules in the same organelle. The clinical symptoms of mitochondrial diseases and the degree of their manifestation directly depend on the number of mutant mtDNA molecules in the cell. The possible way to reduce adverse effects of the mutation is by shifting the level of heteroplasmy towards the wild-type mtDNA molecules. Using this idea, several gene therapeutic approaches based on TALE and ZF nucleases have been developed for this purpose. However, the construction of protein domains of such systems is rather long and laborious process. Meanwhile, the CRISPR/Cas9 system is fundamentally different from protein systems in that it is easy to use, highly efficiency and has a different mechanism of action. All the characteristics and capabilities of the CRISPR/Cas9 system make it a promising tool in mitochondrial genetic engineering. In this article, we demonstrate for the first time that the modification of gRNA by integration of specific mitochondrial import determinants in the gRNA scaffold does not affect the activity of the gRNA/Cas9 complex in vitro.

The task of the objects identification, counting, and measurement is a huge part of scientific investigations and technological applications. Automated methods using traditional processing such as segmentation, edge detection, and so on represented by available software (e.g. CellProfiler) are not flexible, can be used only with images of high-quality, and in addition require setting a part of parameters by hand. This contribution presents the applying the deep learning method for recognition of HeLa cells expressing green fluorescent protein (EGFP) automatically. We used Cascade Mask R-CNN neural networks which has a ResNeXt backbone and deformable convolutional networks layers. Training dataset contained seven pictures with 5754 labeled cells. Three images with 2469 labeled cells were used as test-dataset. The trained neural network showed mAP=0.4.

For medical genetic counseling, estimating the chance of a child being born with chromosome abnormality is crucially important. Cytogenetic diagnostics of parents with a balanced karyotype are a special case. Such chromosome rearrangements cannot be detected with comprehensive chromosome screening. In the current paper, we consider chromosome diagnostics in two cases of chromosome rearrangement in patients with balanced karyotype and provide the results of a detailed analysis of complex chromosomal rearrangement (CCR) involving three chromosomes and a small supernumerary marker chromosome (sSMC) in a patient with impaired reproductive function. The application of fluorescent in situ hybridization, microdissection, and multicolor banding allows for describing analyzed karyotypes in detail. In the case of a CCR, such as the one described here, the probability of gamete formation with a karyotype, showing a balance of chromosome regions, is extremely low. Recommendation for the family in genetic counseling should take into account the obtained result. In the case of an sSMC, it is critically important to identify the original chromosome from which the sSMC has been derived, even if the euchromatin material is absent. Finally, we present our view on the optimal strategy of identifying and describing sSMCs, namely the production of a microdissectional DNA probe from the sSMC combined with a consequent reverse painting.