Molecular Genetic Methods Research Articles

Наследственные гипомагниемии у детей: патогенез, клиническая картина и подходы к лечению

Hypomagnesaemias are the diseases characterized by the reduced level of magnesium in the body. The first mention of them appeared in the early XX century when cases in children were initially described as conditions accompanied by painful twitching in the muscles, weakness and heart rhythm disturbances, the reasons for which remained unknown for a long time. The scientific research in recent decades has only made it possible to decipher the diseases’ pathogenesis mechanisms. Primary hypomagnesaemias are hereditary monogenic diseases in which, as a result of a violation of magnesium metabolism in the body, its concentration decreases and the main clinical manifestations of the disease develop. The clinical picture of hypomagnesemia in children can be diverse and nonspecific. Early symptoms are manifested in the form of short-term involuntary painful contractions of muscles or muscle groups, weakness, cardiac arrhythmia and fatigue. As the disease progresses, more rather serious symptoms such as stunted growth, psychomotor development and epileptic seizures may occur. The severity of clinical manifestations of hereditary hypomagnesaemias correlates with the degree and duration of magnesium deficiency: from general weakness and crumpy syndrome to the development of depression of consciousness and in some cases of an unfavorable outcome. The main methods for confirmatory diagnosis of genetically determined hypomagnesaemias are molecular genetic methods. Authors represent bibliographical review and recent updates on known primary hypomagnesaemias in terms of mechanisms of pathogenesis and clinical presentation as well as their own familial case description of hypomagnesaemia in children.

Molecular tests for hybridization analysis in the diagnosis of drug susceptibility of <i>M. Tuberculosis</i> to fluoroquinolones and injectable drugs

The paper presents data on testing the drug susceptibility of clinical isolates of M. tuberculosis (MTB) to fluoroquinolones and injectable drugs using molecular genetic tests and phenotypic methods. We demonstrated the diagnostic capabilities of the hybridization technologies «TB-TEST and GenoType MTBDRsl v.2 to detect the genetic determinants of resistance in MTB isolates with multidrug and extensively drug resistance (MDR, XDR), as well as in MBT with pre-XDR.

Disorders of carbohydrate metabolism and candidate genes for the pathophysiology of polycystic ovary syndrome. A literature review

Polycystic ovary syndrome is a common heterogeneous disease with metabolic disorders. In the last decade, the study of polycystic ovary syndrome pathogenesis has been associated with the modern development of molecular genetics, transcriptomics, and sequencing methods. Numerous studies have shown that the study of genetic markers and epigenetic changes in metabolic disorders, oxidative stress, chronic inflammation, and mitochondrial dysfunction in polycystic ovary syndrome is an important direction in the pathogenesis and etiology of the disease. The aim of this literature review was to describe candidate genes involved in the pathophysiology of polycystic ovary syndrome and associated with disorders of carbohydrate metabolism according to modern domestic and foreign literature over the past five years. The candidate gene data presented were assessed based on the main aspects of polycystic ovary syndrome pathophysiology, namely, metabolic dysfunction, androgen and gonadotropin imbalance, and inflammation. The insulin genes (variable number of tandem repeats), such as INS-VNTR, IRS-1, IRS-2, and INSR, adiponectin and calpain-10 genes, as well as CY1A1, CYP11A1, PON1, DENND1A and TCF7L2 genes are associated with metabolic disorders in polycystic ovary syndrome. Genetic variants of genes involved in regulating the expression and mechanism of action of insulin, as well as its receptors and substrates (IRS-1, IRS-2, INSR), have been suggested as possible factors involved in the development and severity of the clinical and metabolic manifestations of polycystic ovary syndrome. The presented data on PPARγ gene (and its coactivator PGC-1α) expression levels in women with polycystic ovary syndrome revealed the presence of PPARγ gene polymorphisms associated with insulin resistance. Thus, the data presented in this review from genome-wide association studies (GWAS) and the study of candidate genes showed that numerous pleiotropic effects cause carbohydrate metabolism disorders in polycystic ovary syndrome. The study of genetic markers and epigenetic changes in the development of metabolic disorders, oxidative stress, chronic inflammation, and mitochondrial dysfunction in polycystic ovary syndrome is an important direction in the pathogenesis of the disease.

Radiomic Characteristics of Different T<sub>1</sub> Breast Cancer Biotypes

Background: Breast cancer (BC) occupies a leading position among my oncological diseases detected in women. Identification and search for predictors of malignant neoplasms using radiation and molecular genetic methods of research allows timely diagnosis and treatment, which improves the prognosis for breast cancer. Purpose: To identify a correlation between the molecular subtype of a breast cancer tumor at an early clinical stage and the patterns of the mammographic method. Methods: A prospective, single-center study of 363 patients diagnosed with breast cancer followed up during 2021. X-ray mammography in two projections, ultrasound-guided trephine biopsy for histological verification, and immunohistochemical (IHC) analysis to determine molecular subtypes were performed. Results: There were statistically significant differences in age between subtypes luminal A, luminal BHER2+ (p < 0.001) and triple negative (p = 0.037), luminal B, luminal BHER2+ (p = 0.001) and triple negative (p = 0.046), luminal BHER2+ and nonluminal HER2+ (p = 0.002), between nonluminal HER2+ and triple negative subtype (p = 0.034). When comparing the structure of radiological density, statistically significant differences were revealed between the subgroups luminal B, luminal BHER2+ (p = 0.010) and triple negative (p = 0.010), between luminal A and triple negative subtypes (p = 0.010). When comparing the leading mammographic symptom (p < 0.001), radiological contours of the formation (p < 0.001), the density of pathological changes (p < 0.001), the size, the newly detected pathological process (p < 0.001) statistically significant differences were also found in the subgroups. A division into groups according to the size of pathological changes within the biotypes was noted, where the aggressive phenotypes of the triple negative subtype (p = 0.001), non-luminal HER2+ (p = 0.02) and luminal B (p = 0.02), in contrast to luminal A, were manifested by a greater extent. the maximum linear size of the tumor. A symptom of nipple retraction (p = 0.048) was described, which was not characteristic of triple negative and non-luminal HER2 cancer. Conclusions: Visualization features of differences in the radiological manifestation of breast cancer of different biological subtypes up to 20 mm can be predictors of molecular subtypes. Pathological verification and IHC study remain a mandatory study, but it may be necessary to conduct an X-ray histological correlation before starting treatment and, if obvious discrepancies are detected, repeat the IHC analysis from the surgical material.

Organization for laboratory diagnosis of SARS-COV-2 in the conditions of an anti-tuberculosis institution

Introduction. In connection with the spread of a new coronavirus infection COVID-19 since 2020, measures have been taken in the city of Moscow to widely diagnose the causative agent of COVID-19. A laboratory PCR department was opened in the State Budgetary Healthcare Institution «MNPC for the fight against tuberculosis of the DZM», taking into account the available material and human resources. Purpose of the study: To generalize the experience of organizing the laboratory service of an anti-tuberculosis institution in the city of Moscow during the spread of a new coronavirus infection. Results. Human and material resources have made it possible to maintain the quality and availability of laboratory diagnostics of biological material from patients receiving treatment at the Moscow Regional Research and Practical Center for Combating Tuberculosis, DZM, during the COVID-19 pandemic. In the period 2020-2022 in total, 90,102 studies of biological material (smears) were carried out by RT-PCR in order to detect COVID-19 RNA. Starting from June 2020 to the present, the PCR department of the KDL Clinic 2 has been studying the direct detection of Covid-19 RNA in biological material using molecular genetic methods based on reverse transcription and polymerase chain reaction (RT-PCR) and the identification of specific immunoglobulin classes IgM and IgG to coronavirus antigens in blood serum (plasma) by the method of immunochemiluminescent analysis (ICLA) of both persons receiving treatment at the State Budgetary Institution of Healthcare Center for Combating Tuberculosis Moscow, and employees.

Assessment of the transcriptional activity of cyclin genes in gastric tissues of patients infected with Helicobacter pylori

The article presents the result of the authors' own experience in the study of transcriptional activity of cyclin genes in gastric tissue samples from patients diagnosed with gastric malignant neoplasm against the background of confirmed Helicobacter pylori infection. Based on the obtained data, a molecular-genetic method of assessing the pathophysiological state of gastric tissues on the basis of complex analysis of the level of transcriptional activity of cyclin genes is proposed.

Survivability and proliferation of microorganisms in bentonite with implication to radioactive waste geological disposal: strong effect of temperature and negligible effect of pressure

As bentonite hosts a diverse spectrum of indigenous microorganisms with the potential to influence the long-term stability of deep geological repositories, it is essential to understand the factors influencing microbial activity under repository conditions. Here, we focus on two factors, i.e., temperature and swelling pressure, using a suspension of Cerny Vrch bentonite to boost microbial activity and evaluate microbial response. Suspensions were exposed either to different pressures (10, 12 and 15 MPa; to simulate the effect of swelling pressure) or elevated temperatures (60, 70, 80 and 90 °C; to simulate the effect of cannister heating) for four weeks. Each treatment was followed by a period of anaerobic incubation at atmospheric pressure/laboratory temperature to assess microbial recovery after treatment. Microbial load and community structure were then estimated using molecular-genetic methods, with presence of living cells confirmed through microscopic analysis. Our study demonstrated that discrete application of pressure did not influence on overall microbial activity or proliferation, implying that pressure evolution during bentonite swelling is not the critical factor responsible for microbial suppression in saturated bentonites. However, pressure treatment caused significant shifts in microbial community structure. We also demonstrated that microbial activity decreased with increasing temperature, and that heat treatment strongly influenced bentonite microbial community structure, with several thermophilic taxa identified. A temperature of 90 °C proved to be limiting for microbial activity and proliferation in all bentonite suspensions. Our study emphasizes the crucial role of a deep understanding of microbial activity under repository-relevant conditions in identifying possible strategies to mitigate the microbial potential within the deep geological repository and increase its long-term stability and safety.

The use of ITS markers for identification of birch species from section Apterocaryon

Betula nana, B. humilis are shrub birch forms of the section Apterocaryon (dwarf birches), widespread in the Northern Hemisphere. The species are found on marsh, tundra and bald soils, often growing together on sphagnum bogs. There were suggestions about the hybridization of the diploid B. nana and B. humilis with the tetraploid downy birch B. pubescens from the section Betula (white birches). Attempts were made to study the presence and intensity of hybridization of these species using molecular genetic methods. The aim of the study is to investigate the genetic polymorphism of B. nana and B. humilis in the Middle Urals based on ITS markers of nuclear ribosomal DNA. These markers are for the first time studied in shrub birch forms on the Russian Federation territory. It is shown that the ITS 1-2 region is informative and makes it possible to differentiate closely related birch species B. humilis, B. nana growing in the Middle Urals. Species were characterized by individual ribotypes; B. nana samples had 5 ribotypes, and B. humilis samples had 2 ribotypes. Populations of B. nana showed greater variability in this region compared to B. humilis, which may indicate a higher adaptive potential of dwarf birch, as well as the absence of significant changes in the population size of this species during the last Quaternary glaciation.

Analysis of SMN1, NAIP and GTF2H2 gene status in correlation with spinal muscular atrophy

Introduction. Spinal Muscular Atrophy (SMA) is a genetic disorder caused by the loss of the survival motor neuron (SMN1) gene in over 95% of cases. Additionally, mutations in genes associated with the SMA chromosomal region can influence disease progression. Aim: To analyze the status of the NAIP and GTF2H2 genes in correlation with SMA. Material and methods. The study included 105 patients suspected for SMA of which 50 with confirmed with SMA and 55 without causative deletions, and 107 healthy, unrelated individuals. The molecular genetics methods used were mPCR, PCR-RFLP and MLPA. Results. From 105 patients, 50 were confirmed with SMA. In this group were identified in 8 patients (16%) with a homozygous deletion of exon 5 of the NAIP gene, 4 patients (8%) had a heterozygous status, and 2 (4%) had duplications. In the rest of the patients (55), in which deletions of SMN1 exon 7 were not identified, homozygous deletion of exon 5 of the NAIP gene was established in one patient (2%), 3 patients (5%) had duplications of exon 5 of the NAIP gene, and one patient had 5 copies of the NAIP gene. In the 107 healthy controls, one patient (1%) was identified with a deletion of exon 5 of the NAIP gene. None of the patients with combined deletions of SMN1 and NAIP had deletions in GTF2H2. Conclusions. The frequency of deletions in the NAIP gene was found to be higher in the SMA patient group compared to the control group. Thus, a significant relationship was identified, the P value being <0.00001. The significance threshold was set at p<0.05. The genetic patterning of genes associated with SMA is an important aspect in the study of molecular pathophysiology and assessment of disease prognosis, especially in the approach to gene therapies.

Precision medicine in infectious diseases

Infectious medicine faces avariety of challenges, such as the increase in antibiotic resistance and the emergence and spread of infectious diseases fueled by climate change and globalization. Precision medicine can provide solutions to many of these challenges. Since an untargeted request for diagnostic tests can lead to test results without clinical relevance, which can increase the use of non-indicated antibiotics, the principle aimed at is: targeted diagnostics (the right test) and consideration of patient characteristics (the right person) to optimize management (the right action). At the same time, one must always decide whether empirical therapy must be immediately initiated, even if the results of the initiated diagnostics are not yet available. In addition, many new diagnostics as well as therapies have recently been developed for the rapid detection and more specific treatment of bacterial infections. Molecular genetic methods, which offer more rapid results than classical bacterial cultures, are gaining ground as new diagnostics. New therapeutics such as bacteriophages, antibodies or antibacterial peptides allow increasingly precise treatment of certain bacterial infections. Precision medicine will also play an increasingly important role in infectious medicine in the future.

Etiology of community-acquired pneumonia in children and adults based on 2015–2022 bacteriological and molecular genetic studies

The data on etiology of community-acquired pneumonia (CAP) remain contradictory, which is often assessed only according to bacteriological studies coming to a conclusion about the low frequency of major main pathogenStreptococcus pneumoniaеisolation. At the same time, there are very few observations of parallel examination of patients with CAP simultaneously by bacteriological and molecular genetic methods. There are no observations on the etiology of CAP in the long-term dynamics. The aim of the work is to analyze the etiology of CAP in children and adults based on 2015–2022 bacteriological and molecular genetic studies. The etiology of pneumonia was assessed using the data on posterior pharyngeal wall washes from 418 children and 483 adults hospitalized with a primary diagnosis of CAP at two large Perm medical organizations in 2015–2022. The samples were examined by the bacteriological method and polymerase chain reaction using commercial test systems with the amplifier “CFX96” (Bio-Rad, USA). The results showed thatS. pneumoniaе, despite implementing decreed vaccinations against pneumococcal infection, it remains the leading causative agent of CAP in both children and adults. The 2015–2022 frequency of pneumococcal discharge according to bacteriological studies was 9.7 per 100 examined, according to molecular genetic studies — 48.5 per 100 examined among children and adults in the study area. According to 2021–2022 vs 2015–2017 bacteriological studies, the frequency ofStaphylococcus aureusexcretion among children increased significantly, andS. pneumoniaеincreased among adults. According to the results of molecular genetic studies among children and adults, no significant changes in the etiology of CAP were found. In 2021–2022, SARS-CoV-2-positive vs SARS-CoV-2-negative patients with CAP infected had higher rate of isolatedS. aureus,KlebsiellapneumoniaеandHemophilus influenzae, i.e., those pathogens whose number increased in the long-term follow-up observations.

Testing a set of SSR markers to obtain the genetic profiles of common plum and cherry plum varieties and to assess the genetic similarity between hybrids obtained in remote crossing

Common plum (Prunus domestica L.) is one of the fruit crops that are particularly difficult to study genetically. Unlike most other species of the genus Prunus, it is a hexaploid species (2n=48) originating from complex interspecific hybridization, and the question of its origin is yet to be conclusively resolved. Presently, several studies are available that examine the phylogeny and systematics of the genus Prunus in general and specifically the speciation of Prunus domestica L., with the use of molecular genetics methods. Although the SSR marker method is widely adopted in the practice of studying the most important stone fruit crops, the polymorphism of SSR loci in Prunus domestica L. is rarely studied in the gene pool. The present study examined the effectiveness of 22 microsatellite (SSR) markers using common plum and cherry plum varieties bred at the Federal Horticultural Center for Breeding, Agrotechnology, and Nursery in order to obtain the genetic profiles of these varieties and develop a variety passport. In addition, this set of markers was used to evaluate the genetic diversity and genetic similarity between the varieties of common plum and cherry plum, as well as remote hybrids obtained using embryo rescue, from the collection of the Federal Horticultural Center for Breeding, Agrotechnology, and Nursery. The screening results obtained for a set of 22 SSR markers using 10 varieties and 19 hybrid specimens revealed a low level of polymorphism detected by this set and a small number of shared alleles. This set of markers was found to be inadequate to fully evaluate our sample in terms of allelic diversity, as well as genetic similarity between parental and hybrid forms. The proportion of unique genotypes that can be identified using these markers amounts to less than 50 %. The large number of overlapping fragment sizes prevents the generation of individual genetic profiles for the varieties. It was found necessary to apply and test additional markers, as well as to develop multiplex sets on their basis, which will form the basis for routine genotyping of varieties. For a more accurate and reliable estimation of fragment lengths and allele sharing, further analysis should be performed using capillary electrophoresis. The work was carried out at the Laboratory of Reproductive Biotechnology of the Federal Horticultural Center for Breeding, Agrotechnology and Nursery in 2022.

Microbiological and molecular genetic characterization of Staphylococcus aureus and Staphylococcus pseudintermedius

Coagulase-positive staphylococci are an important infectious agentcausing numerous infections in animals. Staphylococcus aureus andStaphylococcus pseudintermedius share a number of similar cultural andbiochemical characteristics, which makes their differentiation difficult.Since these species have different zoonotic potential, it is advisable todevelop rapid and specific schemes for species differentiation of pathogens.We have studied the cultural and biochemical characteristics ofStaphylococcus spp. isolated from dogs, cats and cows. In total, 103halophilic coccal cultures were isolated from companion animals and45 from cows, of which 55 and 30 cultures were coagulase-positivestaphylococci, respectively. The reactions that can be used to differentiateS. pseudintermedius and S. aureus were studied. Growth inhibition zonesaround the disk with polymyxin B antibiotic for S. pseudintermediuswere statistically higher (p<0.001) than for S. aureus. The determinationof acetone production to differentiate between these pathogens hasless specificity, as 30% of S. pseudintermedius showed a false-positivereaction. The belonging of two isolates to the species Staphylococcuspseudintermedius was confirmed by MALDI-TOF.The virulence of staphylococci is due to the presence of genesthat regulate the synthesis of various pathogenicity factors and causeantibiotic resistance. Molecular genetic methods can detect the presenceof gene specificity and help to assess the risk of a particular strain causinginfection. Using classical and real-time PCR, the mecA gene was detectedin 8 S. aureus strains and 1 S. pseudintermedius strain that showedphenotypic resistance to methicillin. The pathogenicity genes lukF andsiet were present in 100%, and the lukS gene in 90% of the studiedStaphylococcus pseudintermedius.The study highlights a number of aspects of the diagnosis anddifferentiation of coagulase-positive staphylococci. The possibility ofusing the Neonatal FAST well D-ONE microculture system for use inveterinary laboratories was also studied. The data obtained can be usedto develop methodological approaches to the identification of pathogenicstaphylococci using a combination of different methods. Key words: S. pseudintermedius, resistance to methicillin,pathogenicity genes, MALDI-TOF MS.

The use of DNA markers in raspberry (Rubus L.) research: a review

Raspberry (Rubus L.) is one of the most common berry crops in horticulture. It is a valuable food product for humans and a raw material for food processing companies. The assortment of raspberries in Ukraine includes more than 30 varieties. Modern breeding and genetic programs are aimed at expanding genetic diversity and creating new raspberries varities. Molecular genetic methods are increasingly being used in both fundamental and applied research of Rubus species. This article presents an overview of the main types of molecular markers used to study genetic polymorphism of Rubus species. Out of the whole variety of available DNA markers, such molecular methods of analysis as RAPD, RFLP, AFLP, ISSR, SSR and SNPs have proved to be the most effective in solving problems related to genotypes, population polymorphism, genetic mapping, and phylogenetic studies of raspberries. Their high efficiency is associated with increased resolution, reproducibility, high informativeness, the possibility of analysis automatization, speed, simplicity and availability. These markers are a convenient tool for genomic selection and research of genetic diversity of not only the genus Rubus representatives, but also of all living organisms. As of retrotransposon markers, which make up the main part of the eukaryotes genome, there are few scientific papers on their use for the study of representatives of the genus Rubus, unlike other crops. Significant progress in raspberry breeding is associated with the development of modern sequencing technologies. Whole-genome sequencing (WGS) allows simultaneous generation of a large number of SNP markers that are used to create genetic maps, identify pathogen resistance genes, map economically useful traits etc. Key words: Rubus, raspberry, DNA markers, polymorphism, selection.

Genetic sexing of subadult skeletal remains

When subadult skeletons need to be identified, biological sex diagnosis is one of the first steps in the identification process. Sex assessment of subadults using morphological features is unreliable, and molecular genetic methods were applied in this study. Eighty-three ancient skeletons were used as models for poorly preserved DNA. Three sex-informative markers on the Y and X chromosome were used for sex identification: a qPCR test using the PowerQuant Y target included in PowerQuant System (Promega), the amelogenin test included in ESI 17 Fast STR kit (Promega), and a Y-STR amplification test using the PowerPlex Y-23 kit (Promega). Sex was successfully determined in all but five skeletons. Successful PowerQuant Y-target, Y-amelogenin, and Y-chromosomal STR amplifications proved the presence of male DNA in 35 skeletons, and in 43 subadults female sex was established. No match was found between the genetic profiles of subadult skeletons, and the elimination database and negative control samples produced no profiles, indicating no contamination issue. Our study shows that genetic sex identification is a very successful approach for biological sexing of subadult skeletons whose sex cannot be assessed by anthropological methods. The results of this study are applicable for badly preserved subadult skeletons from routine forensic casework.

Analysis of coxsackievirus A6 circulation in the territories of the Far Eastern Federal district of the Russian Federation in 2014–2019

Introduction. Molecular epidemiological monitoring of enterovirus infection (EVI) in the territories of the Russian Federation showed that coxsackievirus A6 (CVA-6) had been one of the most prevalent types of enteroviruses that circulated among the country population during last years and had caused majority of EVI outbreaks.
 Objective — to evaluate coxsackievirus A6 circulation in the territories of the Far Eastern Federal district (FEFD) in 2014–2019 utilizing methods of molecular genetics.
 Materials and methods. RT-PCR was employed to detect enterovirus RNA in biological material collected in 9 territories of the FEFD . In order to establish enterovirus types, amplification of positive samples was carried out to detect nucleotide sequence fragments of the VP1 and VP2 genes. Molecular genetic analysis of the Far Eastern CVA-6 strains was based on detection of nucleotide sequences of VP1 and 3Dpol gene fragments. Phylogenetic trees were constructed by the means of Bayesian phylogenetic methods.
 Results. Total 1773 nucleotide sequences of 43 types of non-polio enteroviruses were obtained in 2014-2019. Majority of the sequences belonged to coxsackievirus A6 (524; 29.5%). In the years of the highest CVA-6 detection an increase in EVI incidence as well as EVI outbreaks were observed in the territories of FEFD. The most prevalent manifestations of EVI caused by CVA-6 in FEFD were herpangina and exanthemic forms. Phylogenetic analysis showed that Far Eastern strains of CVA-6 during in the analyzed period of time belonged to D3 subgenotype that is dominant in the world. The circulation of several recombinant forms of CVA-6 (RF-A, -H, -L, -N, -R) was also registered.
 Conclusion. The genetic diversity of CVA-6 strains circulating in the territories of the FEFD in 2014–2019 revealed in this study requires further investigation in order to obtain new knowledge about the CVA-6 molecular epidemiology and improve the enterovirus surveillance system.

Chinese experts' consensus guideline on preimplantation genetic testing of monogenic disorders.

Recent developments in molecular biological technologies and genetic diagnostic methods, accompanying with updates of relevant terminologies, have enabled the improvements of new strategies of preimplantation genetic testing for monogenic (single gene) disorders (PGT-M) to prevent the transmission of inherited diseases. However, there has been much in the way of published consensus on PGT-M. To properly regulate the application of PGT-M, Chinese experts in reproductive medicine and genetics have jointly developed this consensus statement. The consensus includes indications for patient selection, genetic and reproductive counseling, informed consent, diagnostic strategies, report generation, interpretation of results and patient follow-ups. This consensus statement serves to assist in establishment of evidence-based clinical and laboratory practices for PGT-M.

Diagnostics of combined hereditary cardiovascular pathology in a teenager

Dilated cardiomyopathy is one of the leading causes of heart failure in children with a variety of clinical characteristics. A 12-year-old patient with dilated cardiomyopathy and heterozygous familial hypercholesterolemia, who underwent surgical treatment for implantation of the Abbott HeartMate III Left Ventricular Assist Device as a bridge to donor heart transplantation, is presented. Establishing an accurate diagnosis in the presented observation became possible thanks to the use of a modern molecular genetic method — whole genome DNA testing by next generation sequencing (NGS). The description of this case will help expand the range of possible clinical manifestations of dilated cardiomyopathy and reminds us of the importance of dynamic monitoring of children with cardiac arrhythmias.

Fine-needle aspiration biopsy possibilities in studying the molecular genetic landscape of breast tissue

Background. Core biopsy of the breast is currently considered to be the standard method of obtaining material for morphological and molecular genetic methods. Unfortunately, this method is associated with a number of problems, primarily the risk of complications (bleeding, pneumothorax) and discomfort during manipulation.Aim. To analyze transcriptional signatures of breast tissue samples obtained by fine-needle aspiration biopsy. Materials and methods. Using reverse transcriptase polymerase chain reaction, we studied the mRNA expression level of 60 target genes in 60 samples obtained by fine-needle aspiration biopsy and in 60 corresponding formalin-fixed paraffin-embedded (FFPE) surgical specimens of breast. Samples were obtained from the tumor, adjacent tissue, the so-called tumor bed and formally normal tissue at a distance from the primary lesion.Results. A comparative analysis of transcriptional signatures in samples obtained by fine-needle aspiration biopsy and FFPE specimens (120 samples in total) reveled the strongest correlations between transcriptional signatures in biopsy samples and FFPE specimens of tumors. Very strong correlation in tumor samples was established for one gene (CTSL2); strong for 18 genes (MKI67, MYBL2, NAT1, PTEN, TPX2, PTTG1, UBE2T, CCNB1, ESR1, CCND1, MYC, SCGB2A2, MIA, TRAC, FGFR4, ANLN, GSTM1, PRLR); averages for 28 genes (PGR, AURCA, KRT5, FOXA1, SFRP1, EMSY, EXO1, PAK1, KIF14, ERBB2, MMP11, BCL2, BAG1, TMEM45B, BIRC5, CD274/PDL1, ZNF703, TYMS, CCNE1, TPT1, TMEM45A, BRCA1, BRCA2, ESR2, STS, TNFSF11/RANKL, TNFRSF11B/OPG, TNF); weak for 4 genes (GRB7, EGFR, PGRMC1, CYP19A). The presence of correlations between transcriptional signatures in biopsy samples and FFPE specimens can be established in case of sufficient material corresponding to sample intake control (SIC) ≥5 lg for B2M gene.Conclusion. The ability to conduct molecular genetic research on small samples of breast tissue makes it possible to obtain the material using the most minimally invasive method. And this, in turn, expands the possibilities of “genetic monitoring” of cancer, as well as the possibility of more accurate assessment the risks of malignant tumor development in the settings of benign conditions in women with fibrocystic disease and increased mammographic density.

The problem of toxoplasmosis in outpatient practice. Part III. Toxoplasmosis in pregnants and congenital toxoplasmosis

Congenital toxoplasmosis is a consequence of acute toxoplasmosis in a pregnant woman. When assessing the risk of сongenital toxoplasmosis during pregnancy, methods for determining specific antibodies, the genetic material of the pathogen (polymerase chain reaction method), immunoglobulins G avidity are used. Molecular genetic methods have the greatest diagnostic value in the diagnosis of сongenital toxoplasmosis in children of the first year of life. Drug prevention of сongenital toxoplasmosis in Russia can be implemented with the use of spiramycin, antenatal therapy is impossible, and postnatal therapy is limited by the list of available antiprotozoal drugs. For the prevention of сongenital toxoplasmosis, the awareness of the population about the ways and factors of transmission of the pathogen is of primary importance.