Культуры реснитчатых клеток для диагностики первичной цилиарнои дискинезии

Primary ciliary dyskinesia (PCD) is a hereditary autosomal recessive disease that results in a defect in the ultrastructure of epithelial cilia. To date, there is no single diagnostic test for PCD, so the diagnosis is based on the results of multiple tests, such as DNA diagnostics, assessment of nasal nitric oxide levels, ciliary beat frequency (CBF) in nasal biopsy, ciliary ultrastructure, etc. Diagnosis of PCD can be difficult due to secondary damage to the airway epithelium, leading to undiagnosed or false positive cases.The aim of this work was to review studies on the cultivation of human nasal epithelial cells and subsequent differentiation into ciliated cells for the diagnosis of PCD.Conclusion. In vitro ciliogenesis helps to make a correct diagnosis of PCD while avoiding false positives. There are three different methods of ciliogenesis in vitro: the suspension culture method, the ALI culture method, and the organoid culture method. Each method of ciliogenesis has its own advantages and disadvantages. The ALI culture method is the most widely used. It produces a sufficient number of ciliated cells for diagnosis, which can be maintained in culture for a long time. The obtained cultures of nasal epithelial ciliated cells allow to analyze the ultrastructure of cilia, to evaluate CBF and localization of ciliary proteins, which helps in the diagnosis of PCD.Первичная цилиарная дискинезия (ПЦД) — наследственное аутосомно-рецессивное заболевание, которое приводит к дефекту ультраструктуры ресничек эпителия. На сегодняшний день нет единого метода диагностики ПЦД, поэтому диагноз устанавливается по результатам нескольких анализов, таких как ДНК-диагностика, оценка уровня назального оксида азота, частота биения ресничек (ЧБР) по данным назальной биопсии, ультраструктура ресничек и др. Диагностика ПЦД может быть затруднена из-за вторичных повреждений респираторного эпителия, при этом результат может оказаться отрицательным или ложноположительным.Целью работы явился обзор исследований, посвященных культивированию назальных эпителиальных клеток человека, с последующей дифференцировкой в реснитчатые клетки для диагностики ПЦД.Заключение. Цилиогенез in vitro помогает при установлении правильного диагноза у пациентов с ПЦД, избегая при этом проблем с ложноположительным диагнозом. Существуют 3 различных метода цилиогенеза in vitro: суспензионная культура, ALI-культура и культура органоидов. Каждый метод имеет свои преимущества и недостатки. Метод ALI-культуры является наиболее распространенным, при котором образуется достаточное для диагностики количество реснитчатых клеток, способных длительно поддерживаться в культуре. Полученные культуры реснитчатых клеток назального эпителия позволяют проводить анализы ультраструктуры ресничек, оценивать ЧБР и локализацию цилиарных белков, что помогает при диагностике ПЦД

Cardiomyopathies associated with the DES gene mutations: molecular pathogenesis and gene therapy approaches

Cardiomyopathy (CMP) is a common group of cardiovascular disorders. Genetic (primary) cardiomyopathies are related to abnormalities in more than 100 genes, including the DES gene encoding desmin protein. Desmin is an essential member of the intermediate filaments, ensuring the structural and functional integrity of myocytes. Mutations in the DES gene result in desmin-related cardiomyopathy with progressive course and poor prognosis. By now, specific therapy for cardiomyopathy has not been developed. Existing conservative and surgical treatment modalities target the rate of heart failure progression and sudden cardiac death prevention but have limited efficacy. The development of gene therapy and genome editing could allow for creating effective and specific methods of gene-based therapy for desminopathies. A  number of studies have been published on the use of gene therapy for various genetic cardiomyopathies including those caused by the DES gene mutations, while genome editing has not been used yet. However, promising results have been obtained with CRISPR/Cas9 and TALEN editing systems to correct for “gain-of-function mutations” in some other genes, such as MYBPC3 and PLN. There is also evidence of the possibility to reduce the symptoms of desmin-related cardiomyopathy up to the normal function by knocking out the mutant DES allele, and preserved protein function provided by expression of the normal allele. We believe that genome editing approaches have an open perspective into the development of specific and effective methods to treat desminopathies

The comparative analysis of BCR-ABL/ABL detection by real-time quantitative PCR and automated GeneXpert Dx System in chronic myeloid leukemia patients with major and complete molecular response

Key words: chronic myeloid leukemia, BCR-ABL expression, automated GeneXpert metho

Exome, transcriptome and miRNA analysis don’t reveal any molecular markers of TKI efficacy in primary CML patients

Abstract Background Approximately 5–20% of chronic myeloid leukemia (CML) patients demonstrate primary resistance or intolerance to imatinib. None of the existing predictive scores gives a good prognosis of TKI efficacy. Gene polymorphisms, expression and microRNAs are known to be involved in the pathogenesis of TKI resistance in CML. The aim of our study is to find new molecular markers of TKI therapy efficacy in CML patients. Methods Newly diagnosed patients with Ph+ CML in chronic phase were included in this study. Optimal and non-optimal responses to TKI were estimated according to ELN 2013 recommendation. We performed genotyping of selected polymorphisms in 62 blood samples of CML patients, expression profiling of 33 RNA samples extracted from blood and miRNA profiling of 800 miRNA in 12 blood samples of CML patients. Results The frequencies of genotypes at the studied loci did not differ between groups of patients with an optimal and non-optimal response to TKI therapy. Analysis of the expression of 34,681 genes revealed 26 differently expressed genes (p < 0.05) in groups of patients with different TKI responses, but differences were very small and were not confirmed by qPCR. Finally, we did not find difference in miRNA expression between the groups. Conclusions Using modern high-throughput methods such as whole-exome sequencing, transcriptome and miRNA analysis, we could not find reliable molecular markers for early prediction of TKI efficiency in Ph+ CML patients

P.F508del editing in cells from cystic fibrosis patients.

Development of genome editing methods created new opportunities for the development of etiology-based therapies of hereditary diseases. Here, we demonstrate that CRISPR/Cas9 can correct p.F508del mutation in the CFTR gene in the CFTE29o- cells and induced pluripotent stem cells (iPSCs) derived from patients with cystic fibrosis (CF). We used several combinations of Cas9, sgRNA and ssODN and measured editing efficiency in the endogenous CFTR gene and in the co-transfected plasmid containing the CFTR locus with the p.F508del mutation. The non-homologous end joining (NHEJ) frequency in the CFTR gene in the CFTE29o- cells varied from 1.25% to 2.54% of alleles. The best homology-directed repair (HDR) frequency in the endogenous CFTR locus was 1.42% of alleles. In iPSCs, the NHEJ frequency in the CFTR gene varied from 5.5% to 12.13% of alleles. The best HDR efficacy was 2.38% of alleles. Our results show that p.F508del mutation editing using CRISPR/Cas9 in CF patient-derived iPSCs is a relatively rare event and subsequent cell selection and cultivation should be carried out