There are several forms of monoallelic expression, in which the 50:50 ratio in the expression activity of each of the two copies of the gene is disrupted. This review focuses on the phenomenon of random autosomal monoallelic expression (aRME), a process in which only one of the two alleles is transcribed in a single cell, and the allele choice is not determined by the parental origin. One of the central problems in studying this phenomenon is the differentiation of the two forms of aRME: clonal, in which the expression pattern is mitotically inherited, and stochastic, when the allele choice can change over time due to transcriptional bursting. Distinguishing these events is methodologically challenging: divergent experimental approaches and the lack of standardized detection criteria have produced strikingly contradictory estimates of aRME prevalence. Furthermore, single-cell transcriptomic approaches are particularly susceptible to technical artefacts that can generate false-positive monoallelic calls, complicating the distinction between heritable and transient states. In this review, we critically evaluate the evidence for clonal and stochastic aRME, examine the epigenetic mechanisms proposed to maintain monoallelic expression, and discuss its clinical significance, focusing on the role of monoallelic expression in the penetrance of autosomal dominant diseases (including congenital immune disorders and cardiomyopathy), neurodevelopmental disorders, and cancer progression.

Birt-Hogg-Dubé syndrome (BHDS) is a hereditary cancer syndrome caused by pathogenic variants in the FLCN gene. BHDS is characterized by clinical heterogeneity and similarities with other non-hereditary diseases, which can complicate diagnosis. The aim of our study was to analyze FLCN variants in Russian patients and select the optimal diagnostic approach. We studied 121 unrelated patients suspected for BHDS and 29 of their relatives. Germline variants were analyzed using Sanger sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA). Variant annotation was performed according to the ACMG and AMP recommendations. Pathogenic and likely pathogenic (P/LP) FLCN variants were identified in 20.7% of patients, including six new variants. The distribution of FLCN variants in our cohort was consistent with data obtained from other authors. The mean age of patients with P/LP variants was higher than of those without: 46.91 versus 33.8 years (p < 0.05), suggesting the necessity to apply diagnostic criteria in young patients more carefully. The most common clinical manifestation of BHDS was pulmonary cysts/pneumothorax, while the most informative were alterations involving at least two of three organ systems, which was present in all patients with the P/LP variants, but only in 54% without them (p = 0.001). BHDS diagnostics involves sequencing exons 4-14 of the FLCN gene in patients with proposed clinical criteria. If the result is negative, extensive FLCN deletions are excluded using MLPA, and, in the absence of CNV, WGS is performed.

von Hippel-Lindau syndrome (VHLS) is a hereditary cancer syndrome with CNS hemangioblastomas, clear cell renal carcinoma, pheochromocytoma, retinal angiomas, and a number of other manifestations. VHLS is caused by a mutation in the VHL gene and is inherited in an autosomal dominant manner. However, some cases of VHLS develop de novo, and among them, there are rare patients with a mosaic form of the disease. Genetic testing in mosaic patients is prone to false-negative results due to the low copy number of a mutant allele in DNA isolated from the blood. We describe a case of molecular genetic diagnostics of VHLS in a 39-year-old patient using various methods, including mutation analysis in asynchronous primary tumors and repeated DNA analysis from blood using NGS with high coverage for the mutant position. As a result, the patient was diagnosed with a mosaic form of VHLS caused by the variant c.481C>T (p.Arg161Ter), the proportion of which in the blood DNA was 2%. We also summarized the literature data on the mosaic form of VHLS: the severity of clinical manifestations, the features of differential diagnostics of VHLS with a negative result of routine molecular genetic VHL testing, and specific options of active surveillance and treatment for mutation carriers.

The disruption of the epigenetic mechanisms of gene expression regulation due to the emergence of pathogenic variants in genes-encoding elements of epigenetic machinery leads to the development of chromatinopathies. This group of hereditary diseases includes 179 syndromes, some of which present with overlapping phenotypes. Despite the variety of approaches to molecular diagnostics of chromatinopathies, it is not always possible to establish the molecular diagnosis by traditional methods; thus, the issue of optimizing diagnostic algorithms remains relevant. One of the most rapidly expanding areas of post-genomic molecular diagnostics is episignature detection, which relies on genome-wide DNA methylation analysis. This article aims to represent an original approach to indirect diagnostics of chromatinopathies on the example of Rubinstein-Taybi syndrome 1, which is based on the analysis of the methylation level of a limited set of loci designed to reproduce its classic episignature. In the current study, we apply two methods of targeted quantitative analysis of DNA methylation, which are relatively accessible and can be integrated into diagnostic practice. We demonstrate that Rubinstein-Taybi syndrome 1 episignature may be successfully reduced to a single locus of human genome, and that quantitative bisulfite DNA methylation analysis at this locus allows accurate identification of the Rubinstein-Taybi syndrome 1 patients.

Hereditary papillary renal carcinoma (HPRC) is a rare monogenic hereditary disease in the group of hereditary cancer syndromes. Clinically, HPRC results in the development of multiple papillary renal cell carcinomas of the kidneys in young adults. HPRC is caused by point activating mutations in the MET gene encoding a transmembrane tyrosine kinase receptor. Until now, all detected germline mutations in HPRC patients were missense variants leading to a constitutive activation of the tyrosine kinase domain. We describe, for the first time, unrelated patients with clinical features similar to HPRC and without MET pathogenic missense variants but harboring an extended heterozygous duplication ~101.4 kb in length (chr7:116740252-116841718) in 7q31.2 determined using whole-genome sequencing (WGS). This duplication results in an additional copy of the MET gene fragment, including exons 5-21. The duplicated exons encode most of the receptor domains. According to the American College of Medical Genetics and Genomics (ACMG) criteria, this duplication is classified as variant of uncertain significance (VUS) at present, but it is not excluded that this duplication may represent an activating mutation. Perhaps, further segregation analysis and functional studies will allow us to more accurately resolve the pathogenicity and diagnostic significance of this germline CNV.

Homologous recombination repair deficiency (HRD) is involved in the development of high-grade serous ovarian carcinoma (HGSOC) and its elevated sensitivity to platinum-based chemotherapy. To investigate the heterogeneity of the HRD-positive HGSOC we evaluated the HRD status, including BRCA mutations, genomic scar score, and methylation status of BRCA1/2 genes in 352 HGSOC specimens. We then divided the HRD-positive cohort into three molecular subgroups, the BRCA mutation cohort (BRCA+), BRCA1 methylation cohort (Meth+), and the rest of the HRD+ cohort (HRD+BRCA-Meth-), and evaluated their first-line chemotherapy response, benefit from olaparib, and progression-free survival (PFS). HRD-positive status was detected in 65% (228/352) of samples. The first group, BRCA+, accounted for 45% (102/228) of HRD positive cases and showed the best outcome in platinum therapy (ORR 96%), the highest olaparib benefit (p = 0.006) and the highest median PFS (46 months). The frequency of the second cohort, Meth+, among HRD-positive patients was 23% (52/228). Patients with Meth+ HGSOC showed a significantly poorer outcome, with a median PFS of 19 months, a significantly lower ORR to platinum therapy (84%) and a modest, but not significant, benefit from olaparib maintenance. The third HRD+BRCA-Meth- group accounted for 32% (74/228) of HRD-positive patients and showed an ORR to platinum therapy similar to that of the BRCA+ group (90%), a higher, but not statistically significant, benefit from olaparib and a median PFS of 23 months. In conclusion, Meth+ subgroup had poor outcomes in terms of chemotherapy response, olaparib benefit, and PFS compared to the other HRD+ subgroups, requiring a more thorough follow-up.

Multilocus imprinting disturbances (MLID) is a molecular subtype of imprinting disorders (IDs), in which multiple methylation abnormalities of imprinted regions and genes are observed in combination with polymorphic clinical manifestations, including overlapping phenotypic features of individual imprinting disorders. The causes of MLID are mutations in genes encoding oocyte and zygotic factors of embryo development, which increases the risk of recurrent birth of children with imprinting disorders in mothers carrying such mutations. Due to the need to understand the exact risk for repeated childbirth, it is advisable to diagnose MLID in patients with an ambiguous phenotype and a negative result of studies on individual imprinting disorders, followed by a search for mutations in MLID-associated genes. The purpose of the work is to describe the clinical and epigenetic characteristics of a patient with MLID. A clinical case of a comorbid patient aged 12 years with an established molecular genetic diagnosis of MLID by the method of methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) is presented. The features of the patient’s phenotype allow us to demonstrate the effect of hypomethylation of several differentially methylated regions of imprinted genes on the formation of a polymorphic phenotype, including overlapping signs of individual imprinting disorders, and to assess the difficulty of making an unambiguous clinical diagnosis for this patient. Pronounced clinical polymorphism, negative results of previously conducted molecular genetic studies on certain forms of imprinting disorders allow us to consider the MLID study as a first-line test for the diagnosis of methylation abnormalities in MLID and imprinting disorders.

Despite advances in the diagnosis and treatment of breast cancer (BC), the main cause of deaths is resistance to existing therapies. An approach to improve the effectiveness of therapy in patients with aggressive BC subtypes is neoadjuvant chemotherapy (NACT). Yet, the response to NACT for aggressive subtypes is less than 65% according to large clinical trials. An obvious fact is the lack of biomarkers predicting the therapeutic effect of NACT. In a search for epigenetic markers, we performed genome-wide differential methylation screening by XmaI-RRBS in cohorts of NACT responders and nonresponders, for triple-negative (TN) and luminal B tumors. The predictive potential of the most discriminative loci was further assessed in independent cohorts by methylation-sensitive restriction enzyme quantitative PCR (MSRE-qPCR), a promising method for the implementation of DNA methylation markers in diagnostic laboratories. The selected most informative individual markers were combined into panels demonstrating cvAUC = 0.83 (TMEM132D and MYO15B markers panel) for TN tumors and cvAUC = 0.76 (TTC34, LTBR and CLEC14A) for luminal B tumors. The combination of methylation markers with clinical features that correlate with NACT effect (clinical stage for TN and lymph node status for luminal B tumors) produces better classifiers, with cvAUC = 0.87 for TN tumors and cvAUC = 0.83 for luminal B tumors. Thus, clinical characteristics predictive of NACT response are independently additive to the epigenetic classifier and in combination improve prediction.

Renal cancer (RC) is one of the three most common diseases in oncologic urology. Its accurate diagnosis and prognosis remain difficult and important problems. Some cases of RC are associated with hereditary cancer syndromes and are caused by germline mutations. This review describes monogenic forms of hereditary RC (von Hippel–Lindau syndrome, Birt–Hogg– Dubé syndrome, hereditary leiomyomatosis and renal cell cancer, hereditary papillary renal carcinoma, BAP1 tumor predisposition syndrome) and diseases with several candidate genes (SDH-mutated tumors, tuberous sclerosis complex). Additionally, the review discusses the increased risk of RC in patients with frequent hereditary cancer syndromes predisposing to the development of a wide range of tumor types: Lynch and Li-Fraumeni syndromes. RC in combination with other carcinomas can develop in patients carrying pathogenic mutations in the candidate genes of different hereditary cancer syndromes – multi-locus inherited neoplasia allele syndrome (MINAS) – which is especially important due to the growing role of high-throughput sequencing in practical oncologic genetics. Additionally, guidelines on modern laboratory genetic diagnostics and active surveillance are presented for each syndrome.

Triple-negative breast cancer (TNBC) is the most aggressive molecular subtype, with a poor survival rate compared to others subtypes. For a long time, chemotherapy was the only systemic treatment for TNBC, and the identification of actionable molecular targets might ultimately improve the prognosis for TNBC patients. We performed a genome-wide analysis of DNA methylation at CpG islands on a collection of one hundred ten breast carcinoma samples and six normal breast tissue samples using reduced representation bisulfite sequencing with the XmaI restriction enzyme (XmaI-RRBS) and identified a subset of TNBC samples with significant hypomethylation at the LTB4R/LTB4R2 genes’ CpG islands, including CpG dinucleotides covered with cg12853742 and cg21886367 HumanMethylation 450K microarray probes. Abnormal DNA hypomethylation of this region in TNBC compared to normal samples was confirmed by bisulfite Sanger sequencing. Gene expression generally anticorrelates with promoter methylation, and thus, the promoter hypomethylation detected and confirmed in our study might be revealed as an indirect marker of high LTB4R/LTB4R2 expression using a simple methylation-sensitive PCR test. Analysis of RNA-seq expression and DNA methylation data from the TCGA dataset demonstrates that the expression of the LTB4R and LTB4R2 genes significantly negatively correlates with DNA methylation at both CpG sites cg12853742 (R = −0.4, p = 2.6 × 10−6; R = −0.21, p = 0.015) and cg21886367 (R = −0.45, p = 7.3 × 10−8; R = −0.24, p = 0.005), suggesting the upregulation of these genes in tumors with abnormal hypomethylation of their CpG island. Kaplan–Meier analysis using the TCGA-BRCA gene expression and clinical data revealed poorer overall survival for TNBC patients with an upregulated LTB4R. To this day, only the leukotriene inhibitor LY255283 has been tested on an MCF-7/DOX cell line, which is a luminal A breast cancer molecular subtype. Other studies compare the effects of Montelukast and Zafirlukast (inhibitors of the cysteinyl leukotriene receptor, which is different from LTB4R/LTB4R2) on the MDA-MB-231 (TNBC) cell line, with high methylation and low expression levels of LTB4R. In our study, we assess the therapeutic effects of various drugs (including leukotriene receptor inhibitors) with the DepMap gene effect and drug sensitivity data for TNBC cell lines with hypomethylated and upregulated LTB4R/LTB4R2 genes. LY255283, Minocycline, Silibinin, Piceatannol, Mitiglinide, 1-Azakenpaullone, Carbetocin, and Pim-1-inhibitor-2 can be considered as candidates for the additional treatment of TNBC patients with tumors demonstrating LTB4R/LTB4R2 hypomethylation/upregulation. Finally, our results suggest that the epigenetic status of leukotriene B4 receptors is a novel, potential, predictive, and prognostic biomarker for TNBC. These findings might improve individualized therapy for TNBC patients by introducing new therapeutic adjuncts as anticancer agents.

Epigenetic regulation is hereditary and non-hereditary changes in the expression of a particular gene without any corresponding structural changes in its nucleotide sequence. Genomic imprinting is an epigenetic mechanism for regulating the expression of homologous genes depending on parental origin, i.e., they are expressed monoallelically in the mammalian diploid cell. Being genetically imprinted, only the maternal or only the paternal genome is unable to ensure normal embryonic development. The most studied epigenetic modification, which plays one of the main roles in the maintenance of imprinting processes, is the specific methylation of cytosine in CpG-dinucleotides. All known imprinted genes contain differential DNA methylation regions on homologous parent chromosomes, which are necessary for their monoallelic expression. However, it is now known that not only DNA methylation, but chromatin remodeling, histone modifications, and non-coding RNAs also ensure the proper functioning of imprinted genes in the human body. Structural and functional disturbances of epigenetic mechanisms lead to imprinting diseases.

В основе инвазии и метастазирования опухолевых клеток лежит эпителиально-мезенхимальный переход (ЭМП). При этом состоянии происходит потеря связи между эпителиальными клетками и появляется подвижный фенотип, характерный для мезенхимальных клеток. Важную роль в процессе ЭМП играет белок Е-кадгерин, который устанавливает и поддерживает межклеточные взаимодействия и позволяет эпителиальным клеткам формировать клеточный пласт. Частичная или полная потеря экспрессии E-кадгерина, происходящая при ЭМП, опосредована механизмами, которые блокируют экспрессию активаторов белка и связанных с ним транскрипционных факторов. Напротив, происходит гиперэкспрессия генов его блокаторов SNAIL1/2, ZEB, TWIST1, GRHL2, OVOL1/2 и PRRX1. E-кадгерин задействован в различных онкогенных сигнальных путях, таких как Wnt/β-catenin, Rho GTPase и EGF/EGFR, благодаря чему он способствует развитию многих опухолей, включая рак желудка (РЖ). Важнейшие компоненты эпигенетического контроля экспрессии генов при канцерогенезе - микроРНК и длинные некодирующие РНК - участвуют в регуляции функции Е-кадгерина. Они действуют непосредственно или опосредованно, через многочисленные факторы, контролирующие транскрипцию гена, и таким образом влияют на пролиферацию и метастазирование опухолевых клеток. Этот обзор посвящен роли E-кадгерина в опухолевой прогрессии, а также участию некодирующих РНК в механизмах его экспрессионного контроля при канцерогенезе желудка. Epithelial-mesenchymal transition (EMT) is the basis of tumor invasion and metastasis. It is characterized by the loss of communication between epithelial cells, which lead to the appearance of a motile phenotype of mesenchymal cells. The E-cadherin protein plays an important role in the EMT process, establishing and maintaining intercellular interactions and allowing epithelial cells to form a cell layer. The partial or complete loss of E-cadherin expression that occurs during EMT is mediated by mechanisms that block the expression of activators of this protein and related transcription factors. In contrast, the inhibitors of E-cadherin SNAIL1/2, ZEB, TWIST1, GRHL2, OVOL1/2, and PRRX1 are overexpressed. E-cadherin is involved in various oncogenic signaling pathways such as Wnt/β-catenin, Rho GTPase and EGF/EGFR. Therefore it contributes to many tumors, including gastric cancer (GC). MicroRNAs and long noncoding RNAs as important epigenetic regulators of gene expression in carcinogenesis are also involved in the regulation of E-cadherin function. They act directly or indirectly, through multiple factors controlling gene transcription, and thus influence tumor cell proliferation and metastasis. This review is focused on the role of E-cadherin in tumor progression and the involvement of non-coding RNAs in the mechanisms controlling its expression in gastric carcinogenesis.

The rate of complete pathomorphological response to neoadjuvant chemotherapy (NACT) in triple-negative breast cancer is low and ranges from 30 to 40%. At the same time, to date, there are practically no predictive markers of the effectiveness of NACT for this subtype of tumors. We suggest an approach to the identification of prognostic epigenetic markers of the sensitivity of triple-negative breast tumors to NACT, which can be diagnosed using methylation sensitive PCR. We have selected markers from the XmaI-RRBS genome-wide bisulfite DNA sequencing dataset performed for tumor biopsies obtained from 34 patients before the initiation of NACT and for six samples of normal breast tissue. The patients were divided into groups of responders (complete pathological response) or nonresponders (presence of residual tumor). CpG dinucleotides were identified, the differential methylation of which distinguishes samples of triple-negative tumors with different sensitivity to NACT, and a panel of methylation markers was developed, including regions of CpG islands of the RUSC1/RUSC1-AS1, MXRA5, and ANKRD46 genes, the design of which meets the requirements for the test systems based on methylation sensitive PCR. The estimated diagnostic accuracy of a panel of these three methylation markers is 0.79 (cvAUC = 0.80, 95% CI: 0.79–0.82).

Цель. Изучение изменений в образцах злокачественных опухолей молочной железы статуса метилирования остатков цитозина в составе CpG-островков, моноаллельно метилированных в норме. Методы. Для определения CpG-динуклеотидов, моноаллельно метилированных в опухолевых и нормальных тканях молочной железы, использовали полученные авторами ранее результаты широкогеномного бисульфитного секвенирования Xmal-RRBS. Изменения моноаллельного метилирования в опухолях визуализировали с помощью гистограмм для различных метилотипов молочной железы. Результаты. В геномах умеренно метилированных опухолей нормально моноаллельно метилированные CpG-динуклеотиды имеют тенденцию к почти равновероятному изменению как в неметилированное, так и в полностью метилированное состояния. В геномах гиперметилированных опухолей нормально моноаллельно метилированные CpG-динуклеотиды чаще меняют своё состояние на полностью метилированное. Заключение. Наблюдаемый характер изменений позволяет предположить, что динамика диктуется в умеренно метилированных опухолях процессами, не определяющими её вектор, а в гиперметилированных - дополнительно - направленным процессом, повышающим уровень метилирования CpG-динуклеотидов. По аналогии с ранее опубликованными результатами исследования динамики изменений метилирования импринтированных локусов в опухолях человека, мы предполагаем, что процесс случайного определения вектора изменения характера метилирования опосредован нарушениями копийности участков моноаллельного метилирования ДНК. Причиной равновероятных разнонаправленных изменений моноаллельного метилирования может быть геномный дисбаланс, который может случайным образом приводить к делециям (или приобретенной однородительской дисомии) либо метилированного, либо неметилированного аллеля. Aim: to evaluate changes in the methylation status of cytosine residues in CpG islands, monoallelically methylated in the norm, in samples of malignant breast tumors. Methods. To determine CpG dinucleotides monoallelically methylated in tumor and normal breast tissues, we used the results of Xmal-RRBS genome-wide bisulfite sequencing obtained earlier. Changes in monoallelic methylation in tumors were visualized using histograms for different breast methylotypes. Results. In the genomes of moderately methylated tumors, normally methylated CpG dinucleotides have a tendency to an almost equiprobable methylation change to both the unmethylated and fully methylated states. In the genomes of hypermethylated tumors, normally monoallelically methylated CpG dinucleotides more often change their state to fully methylated. Conclusions. The observed nature of the changes suggests that the dynamics is dictated in moderately methylated tumors by processes that do not determine their vector, and in hypermethylated tumors, additionally, by a directed process that increases the methylation level of CpG dinucleotides. By analogy with the previously published results of a study of the dynamics of changes in methylation of imprinted loci in human tumors, we assume that the process of random determination of the vector of methylation changes is mediated by copy number aberrations at the regions of monoallelic DNA methylation. The reason for the equally probable bidirectional changes in monoallelic methylation may be a genomic imbalance that can randomly lead to deletions (or acquired uniparental disomy) of either the methylated or unmethylated allele.

Our aim was to identify RB1 alterations causing hereditary low penetrance retinoblastoma and to evaluate how the parental origin of an RB1 mutation affects its phenotypic expression. By NGS and MLPA, RB1 mutations were found in 191 from 332 unrelated retinoblastoma patients. Among patients with identified RB1 mutations but without clinical family history of retinoblastoma, 7% (12/175) were found to have hereditary disease with one of the parents being an asymptomatic carrier of an RB1 mutation. Additionally, in two families with retinoblastoma history, mutations were inherited by probands from unaffected parents. Overall, nine probands inherited RB1 mutations from clinically unaffected fathers and five, from mothers. Yet, we gained explanations of maternal "unaffectedness" in most cases, either as somatic mosaicism or as clinical presentation of retinomas in involution, rendering the proportion of paternal to maternal truly asymptomatic mutation carriers as 9:1 (p = 0.005). This observation supports an assumption that parental origin of an RB1 mutation influences the likelihood of developing retinoblastoma. Additionally, our study revealed a relatively high frequency of asymptomatic carriage of the RB1 mutations among the parents of retinoblastoma patients, highlighting the utmost necessity of molecular analysis among the probands' relatives irrespective of their clinical status and family history of retinoblastoma.

Введение. Спорадическая ретинобластома развивается в результате мутаций de novo в обоих аллелях гена RB1 в клетках сетчатки глаза. При спорадической ретинобластоме первоначальная мутация в гене RB1 нередко является мозаичной, то есть образуется в постзиготической ранней эмбриональной клетке, что приводит к неравномерному распределению мутантных клонов между различными тканями организма. Возможность идентифицировать мозаичный вариант мутации в гене RB1 имеет значение как для медико-генетического консультирования, так и для клинического ведения пациентов, поскольку мозаицизм влияет на развитие клинической картины заболевания, риск развития опухоли в другом глазу и других опухолей и на риск передачи мутации следующему поколению. Цель: установить частоту и спектр постзиготических мозаичных мутаций в гене RB1 в выборке больных со спорадической ретинобластомой, определить содержание мутантного аллеля в образцах с мозаицизмом. Метод. Исследование проведено на материале ДНК лимфоцитов крови больных со спорадической ретинобластомой. Скрининг точковых мутаций, малых инсерций/делеций в гене RВ1 осуществляли методом полупроводникового высокопроизводительного параллельного секвенирования (ВПС). Исключение протяженных делеций в гене RВ1 проводили методом MLPA. Для поиска мозаичных мутаций с очень низким содержанием (менее 10%) мутантного аллеля был разработан и проведен углубленный анализ данных ВПС, основанный на биоинформатических и статистических подходах. Для верификации выявленных мозаичных патогенных мутаций использовали секвенирование ДНК по Сэнгеру. Результаты. В исследованной выборке больных со спорадической унилатеральной формой ретинобластомы мозаичные мутации встречаются чаще, чем при спорадической билатеральной форме; различия статистически достоверны. В то же время, частоты мозаичных мутаций с высокой и низкой представленностью мутантных аллелей между группами больных с унилатеральной и билатеральной ретинобластомой достоверно не различаются. Все мозаичные мутации, представлены нуль-аллелями; мозаичных миссенс-мутаций в нашей выборке не обнаружено. Не выявлено мозаичных мутаций в 1-м и 2-м экзонах гена RB1, расположенных проксимальнее альтернативного промотора, импринтинг которого определяет пенетрантность мутаций в зависимости от родительского происхождения мутантного аллеля. Заключение. Применение глубокого ВПС в сочетании с усовершенствованным алгоритмом анализа результатов, направленным на выявление мозаичных мутаций, повышает эффективность ДНК-диагностики ретинобластомы, способствуя совершенствованию медико-генетического консультирования и лечения больных. Background. Sporadic retinoblastoma develops as a result of de novo mutations in both alleles of the RB1 gene. Often in sporadic retinoblastoma, the initial mutation in RB1 is mosaic, that is, it is formed in a postzygotic, early embryonic cell, which leads to an uneven distribution of mutant clones between different tissues of the body. The ability to identify a mosaic variant of a mutation in the RB1 gene is important for both medical genetic counseling and clinical management of patients, since mosaicism affects the development of the clinical picture of the disease, the risk of developing a tumor in the other eye, as well as other tumors, and the risk of mutation transmission to the next generation. Aim: to establish the frequency and spectrum of somatic mosaic mutations in the RB1 gene in patients with sporadic retinoblastoma and to quantify the content of the mutant allele in cases with mosaicism. Methods. The study was carried out on the DNA of blood lymphocytes from patients with sporadic retinoblastoma. Screening of point mutations, small insertions/deletions in the RB1 gene was performed by semiconductor high-throughput parallel sequencing (NGS). Exclusion of gross deletions in the RB1 gene was performed by MLPA. To search for mosaic mutations with a very low representation (less than 10%) of the mutant allele, an in-depth analysis of the NGS data was developed an in-house algorithm based on bioinformatic and statistical approaches. To verify mosaic pathogenic mutations identified with NGS, Sanger sequencing was used. Results. Mosaic mutations were found more common among patients with sporadic unilateral form of retinoblastoma than in those with sporadic bilateral form; the differences are statistically significant. At the same time, the frequencies of mosaic mutations with a high and low representation of mutant alleles between the groups of patients with unilateral and bilateral retinoblastoma did not differ significantly. All mosaic mutations are null alleles; mosaic missense mutations were not found in our patients’ cohort. No mosaic mutations were detected in the 1st and 2nd exons of the RB1 gene, located proximal to the alternative promoter, the imprinting of which determines the penetrance of mutations depending on the parental origin of the mutant allele. Conclusion. The use of deep high-throughput parallel sequencing in combination with an improved algorithm for analyzing the NGS results, aimed at identifying mosaic mutations, increases the efficiency of DNA diagnostics of retinoblastoma, contributing to the improvement of medical genetic counseling and treatment of patients.

Введение. Ретинобластома - злокачественная опухоль детского возраста, причиной которой является биаллельная инактивация гена RB1. Ранняя молекулярно-генетическая диагностика ретинобластомы необходима как для адекватного выбора алгоритма лечения пациента с такой опухолью, так и для медико-генетического консультирования семьи. Цель: охарактеризовать частоту и спектр мутаций в гене RB1 у российских больных с ретинобластомой. Методы. Исследование проведено на материале ДНК лимфоцитов крови, полученном от 492 больных с ретинобластомой. Скрининг точковых мутаций, малых инсерций/делеций в гене RВ1 осуществляли методом полупроводникового высокопроизводительного параллельного секвенирования. Исключение протяженных делеций в гене RВ1 проводили методом MLPA. Результаты. Исследовано 492 неродственных пациента с ретинобластомой, среди которых 38,2% (188/492) с билатеральной формой заболевания и 61,8% (304/492) - с унилатеральной. В группе больных с билатеральной формой ретинобластомы герминальная мутация обнаружена у 96,8% (182/188) пациентов, в группе больных с унилатеральной формой - у 16,4% (50/304). Суммарно в гене RB1 в исследованной группе пациентов обнаружено 339 мутаций: 232 - герминальных и 107 - соматических. Выявлен практически полный спектр молекулярных изменений, включающий нонсенс-мутации - 37,5% (127/339), миссенс-мутации - 5,3% (18/339), мутации, приводящие к сдвигу рамки считывания - 18,9% (64/339), мутации сайтов сплайсинга - 13,9% (47/339) и протяженные делеции - 24,5% (83/339). Выводы. Применение глубокого высокопроизводительного параллельного секвенирования и метода MLPA позволяет эффективно выявлять молекулярно-генетические изменения в гене RB1. Типы мутаций, обнаруженные в исследованной группе, их частота и распределение совпадают с результатами исследователей из других стран. Background. Retinoblastoma is a childhood malignant tumor caused by biallelic inactivation of the RB1 gene. Early molecular genetic diagnosis of retinoblastoma is necessary both for an adequate choice of an algorithm for treating a patient, and for competent medical genetic counseling of the family Objective. To establish the frequency and spectrum of mutations in the RB1 gene in the group of patients with retinoblastoma. Methods. The study was carried out on the DNA of blood lymphocytes from 492 patients with retinoblastoma. Screening of point mutations, small insertions/deletions in the RB1 gene was performed by semiconductor high-throughput parallel sequencing. Exclusion of gross deletions in the RB1 gene was performed by MLPA. Results. 492 unrelated patients with retinoblastoma were studied, including 38.2% (188/492) with bilateral form and 61.8% (304/492) with unilateral form. In the group of patients with bilateral retinoblastoma, germline mutation was found in 96.8% (182/188) patients, and in the group of unilateral patients, in 16.4% (50/304). In total, the RB1 gene in the studied group of patients 339 mutations were found, 232 germline and 107 somatic. An almost complete spectrum of molecular changes was revealed, including nonsense mutations, 37.5% (127/339); missense mutations, 5.3% (18/339); frame shift mutations, 18.9% (64 / 339); splice site mutations, 13.9% (47/339); and large deletions, 24.5% (83/339). Conclusion. The use of deep high-throughput parallel sequencing and the MLPA method allows efficient detection of molecular genetic changes in the RB1 gene. The types of mutations found in the studied group, their frequency and distribution are the same as the results of researchers in other countries.

Cell transmembrane receptors and extracellular matrix components play a pivotal role in regulating cell activity and providing for the concerted integration of cells in the tissue structures. We have assessed DNA methylation in the promoter regions of eight integrin genes, two nidogen genes, and the dystroglycan gene in normal breast tissues and breast carcinomas (BC). The protein products of these genes interact with the basement membrane proteins LAMA1, LAMA2, and LAMB1; abnormal hypermethylation of the LAMA1 , LAMA2 , and LAMB1 promoters in BC has been described in our previous publications. In the present study, the frequencies of abnormal promoter hypermethylation in BC were 13% for ITGA1 , 31% for ITGA4 , 4% for ITGA7 , 39% for ITGA9 , 38% for NID1 , and 41% for NID2 . ITGA2, ITGA3, ITGA6, ITGB1 , and DAG1 promoters were nonmethylated in normal and BC samples. ITGA4, ITGA9 , and NID1 promoter hypermethylation was associated with the HER2 positive tumors, and promoter hypermethylation of ITGA1, ITGA9 , NID1 and NID2 was associated with a genome-wide CpG island hypermethylated BC subtype. Given that ITGA4 is not expressed in normal breast, one might suggest that its abnormal promoter hypermethylation in cancer is non-functional and is thus merely a passenger epimutation. Yet, this assumption is not supported by our finding that it is not associated with a hypermethylated BC subtype. ITGA4 acquires expression in a subset of breast carcinomas, and methylation of its promoter may be preventive against expression in some tumors. Strong association of abnormal ITGA4 hypermethylation with the HER2 positive tumors (p = 0.0025) suggests that simultaneous presence of both HER2 and integrin α4 receptors is not beneficial for tumor cells. This may imply HER2 and integrin α4 signaling pathways interactions that are yet to be discovered.

Введение. Выявление и всесторонняя характеристика новых молекулярных маркеров злокачественных опухолей остаётся актуальной задачей онкологии. Ранее по результатам проведенного авторами широкогеномного скрининга дифференциального метилирования ДНК нормальных и опухолевых тканей молочной железы (МЖ) было выявлено аномальное деметилирование CpG-островка генов LTB4R/LTB4R2 в опухолях относительно нормы. Цель. Настоящее исследование посвящено молекулярной и клинической характеристике аномального деметилирования этого CpG-островка и экспрессии генов LTB4R/LTB4R2 при раке молочной железы (РМЖ). Методы. Для разведочного анализа использовали полученные авторами ранее результаты широкогеномного бисульфитного секвенирования Xmal-RRBS 110 образцов РМЖ и 6 образцов нормальной МЖ. В этой выборке был проведен сравнительный анализ уровней метилирования CpG-динуклеотидов в нормальной и опухолевой ткани 10 образцов трижды негативного (ТН) РМЖ с аномальным деметилированием LTB4R/LTB4R2 и 6 образцов нормальной МЖ с использованием критерия Манна-Уитни. Для подтверждения аномального деметилирования в образцах ТН РМЖ было использовано бисульфитное секвенирование по Сэнгеру. Для изучения корреляции между уровнями метилирования и экспрессии и для оценки прогностической значимости экспрессии генов LTB4R/LTB4R2 использовали данные 3 уровня по измерению метилирования с чипов Illumina HumanMethylation 450K, экспрессии RNA-seq, а также клинические характеристики 731 образцов из проекта TCGA-BRCA. Отличия между кривыми Каплана-Мейера сравнивали с использованием логранкового критерия. Результаты. Определено, что аномальное деметилирование CpG-островка генов LTB4R/LTB4R2 характерно для эпигеномного подтипа умеренно метилированных образцов ТН РМЖ. Определены дифференциально метилированные CpG-динуклеотиды CpG-островка генов LTB4R/LTB4R2; их дифференциальное метилирование подтверждено бисульфитным секвенированием по Сэнгеру. Установлена отрицательная корреляция между уровнем их метилирования и экспрессией генов LTB4R и LTB4R2. Показано, что общая выживаемость достоверно снижена в группе ТН РМЖ с высокой экспрессией LTB4R, а также в группе нормально-подобных опухолей МЖ с низкой экспрессией LTB4R и в группе LumB опухолей МЖ с низкой экспрессией LTB4R2. Различия показателей выживаемости позволяют рассматривать уровни экспрессии LTB4R/LTB4R2 как прогностический маркер при РМЖ, однако следует учитывать, что прогноз зависит от молекулярного подтипа опухоли. Это относится также к использованию уровней экспрессии LTB4R/LTB4R2 в качестве потенциального предиктивного маркера чувствительности таких опухолей к ингибиторам лейкотриеновых рецепторов при проведении клинических испытаний. Background. The identification and comprehensive characterization of new molecular markers of malignant tumors remains an urgent task of oncology. Earlier, according to the results of a genome-wide screening of differential DNA methylation of normal and tumor breast tissues, abnormal demethylation of the CpG island of LTB4R/LTB4R2 genes in tumors relative to norm was revealed. Aim. The present study focuses on the molecular and clinical characterization of abnormal demethylation of this CpG island and expression of the LTB4R/LTB4R2 genes in breast cancer (BC). Methods. For exploratory analysis, we used the previously obtained results of genome wide bisulfite sequencing of breast cancer (BC) and normal mammary gland samples. From this set, 10 samples of triple-negative (TN) breast cancer with abnormal LTB4R/LTB4R2 demethylation and 6 normal breast samples were selected, for which a comparative analysis of CpG methylation levels in cancer vs norm using the Mann-Whitney test was performed. Sanger bisulfite sequencing was used to confirm abnormal demethylation in TN BC samples. The analysis of the obtained electrophoregrams was carried out using the SeqBase software developed by the authors. To validate and assess the level of predictive significance of LTB4R/LTB4R2 gene expression, level 3 data were used to measure methylation from Illumina HumanMethylation 450K arrays, RNA-seq expression, as well as clinical characteristics for 731 samples from the TCGA-BRCA project. Kaplan-Meier curves were compared using a logrank test. Results. Abnormal demethylation of LTB4R/LTB4R2 genes in breast tumors was confirmed by Sanger sequencing. Among samples from the TCGA-BRCA project, a group of TN BC samples with low methylation of LTB4R/LTB4R2 genes was identified. Overall survival was significantly reduced in the TN breast cancer group with high LTB4R expression, as well as in the group of normal-like breast tumors with low LTB4R expression, and in the LumB group overall survival was significantly reduced when tumors demonstrated low LTB4R2 expression. Conclusions. Fine mapping of abnormal demethylation in individual CpG dinucleotides of the LTB4R/LTB4R2 genes will make it possible to design an effective PCR system that may potentially be used to define patients with TN breast cancer that would benefit from leukotriene receptor therapeutic inhibition. Different expression levels of LTB4R/LTB4R2 allow their use as a prognostic marker for breast cancer, but the prognosis directly depends on the molecular subtype of a tumor. This also applies to the use of different levels of LTB4R/LTB4R2 expression as a predictive marker of the sensitivity of such tumors to leukotriene receptor inhibitors in case they enter clinical trials.