Геномный импринтинг – эпигенетический механизм, определяющий и регулирующий экспрессию гомологичных аллелей генов различного родительского происхождения. Нарушения этого механизма приводят к болезням геномного импринтинга (БГИ). Регуляция импринтинга осуществляется не только в пределах близко расположенных кластеров генов, но и посредством взаимодействий в импринтированных генных сетях (ИГС). Эти взаимодействия могут объяснить некоторые наблюдаемые различия в фенотипах различных БГИ и MLID (multilocus imprinting disturbances – мультилокусных нарушений импринтинга, при которых наблюдаются множественные аномалии метилирования импринтированных районов и генов), корреляции между эпигенотипом и фенотипом которых не всегда очевидна. На сегодняшний день описано не менее 20 БГИ у человека, как с самостоятельными, так и с перекрывающимися клиническими признаками, включая малые аномалии развития, врожденные пороки развития, нарушения метаболизма, особенности интеллектуального, моторного, физического развития. Чаще у пациента с определенной БГИ поражается один специфический импринтированный локус, но появляется все больше сообщений о пациентах с MLID. Причинами MLID являются патогенные варианты в генах, кодирующих ооцитарные и зиготические факторы развития эмбриона, такие как NLRP2, NLRP5, NLRP7, KHDC3L, OOEP, PADI6, TLE6, UHRF1, ZFP57, ARID4A, ZAR1, ZNF445, TRIM28. Патогенные варианты этих генов демонстрируют особый способ наследования, поскольку они становятся функционально значимыми только у женщин-носительниц. Они влияют не на здоровье самой носительницы, а на ее репродуктивный прогноз. При генетическом консультировании следует учитывать, что фенотип, обусловленный нарушениями в генах ооцитарных и зиготических факторов развития эмбриона, проявляется только тогда, когда носителями являются женщины. Таким образом, вариант может передаваться по отцовской линии, не вызывая при этом репродуктивных проблем. MLID являются актуальной и активно изучаемой проблемой клинической и молекулярной генетики. Ввиду возможной схожести клинической картины классических БГИ и MLID, пациентам с подозрением на БГИ целесообразно проводить анализ на MLID для установления дополнительных паттернов метилирования импринтированных дифференциально метилированных районов (ДМР). В семьях пациентов с MLID необходимо проводить поиск генетических вариантов в MLID-ассоциированных генах для установления риска повторного рождения детей с БГИ. Исследование MLID-ассоциированных генов может быть актуально для пациенток с привычным невынашиванием беременности, рецидивирующим пузырным заносом и при исследовании абортивного материала без хромосомных аномалий для определения причин прерывания и планирования последующей беременности. Genomic imprinting is an epigenetic mechanism that determines and regulates expression of homologous alleles of genes of different parental origin. Disturbances in this mechanism lead to imprinting disorders (IDs). Imprinting is regulated not only within closely located gene clusters, but also through interactions in imprinted gene networks (IGNs). These interactions may explain some of the observed differences in the phenotypes of various ImpDis and MLID (multilocus imprinting disturbances, in which multiple methylation abnormalities of imprinted regions and genes are observed), where the correlation between the epigenotype and the phenotype is not always obvious. To date, at least 20 IDs have been described in humans, both with independent and with overlapping clinical signs, including minor developmental anomalies, congenital malformations, metabolic disorders, features of intellectual, motor, and physical development. More often, in an individual with a specific ID, one specific imprinted locus is affected, but there are increasing reports of patients with MLID. The causes of MLID are pathogenic variants in genes encoding oocyte and zygotic embryo development factors, such as NLRP2, NLRP5, NLRP7, KHDC3L, OOEP, PADI6, TLE6, UHRF1, ZFP57, ARID4A, ZAR1, ZNF445, TRIM28. Pathogenic variants of these genes exhibit a distinct mode of inheritance in that they become functionally significant only in female carriers. They do not affect the health of the carrier herself, but her reproductive prognosis. When providing genetic counseling, it should be taken into account that the phenotype caused by disturbances in the genes for oocyte and zygotic factors of embryo development appears only when the carriers are women. Thus, the variant can be passed on through the father’s side without causing reproductive problems. MLID is an actively studied problem in clinical and molecular genetics. Due to the possible similarity of the clinical picture of classical ID and MLID, it is advisable for patients with suspected ID to undergo analysis for MLID to establish additional methylation patterns of imprinted DMRs, since in families of patients with MLID it is necessary to conduct medical genetic counseling with a further search for genetic variants in MLID-associated genes, to establish the risk of recurrent birth of children with ID. Also, the study of MLID-associated genes may be relevant for patients with recurrent miscarriage, recurrent hydatidiform mole, and for the study of abortive material, in the absence of chromosomal abnormalities identified in it, to determine the causes of termination and competent planning of a subsequent pregnancy.

Introduction. Male infertility is a common problem in andrology and occurs in 45 % of infertile couples. Some cases of male infertility caused by genetic reasons: point mutations at some monogenic diseases, AZF deletions or a CFTR mutation compounds; reduced fertility is also associated with polymorphic variants of the genes AR and GSTT1/GSTM1. At the same time, increasing amount of data are being published about the role of epigenetic mechanisms (aberrant methylation and imprinting alterations) in defective spermatogenesis. Materials and methods. We have studied 49 sperm samples obtained from unrelated infertile men using polymerase chain reaction, fragment analysis, and sequencing. Results. Five patients were excluded from the initial cohort: one with a repeat length of 29 (CAG) in the first exon of the AR, three with null genotypes in the GSTT1 and GSTM1, and one with the delF508/5T heterozygous compound in the CFTR. Thereafter, methylation of the imprinted gene SNRPN was determined using methyl-specific polymerase chain reaction and bisulfite sequencing. Aberrant SNRPN methylation was detected in 11.4 % of male infertility samples. In total, molecular genetic and epigenetic alterations were determined in 20 % of patients. Conclusions. Obtained data demonstrate a significant proportion of (epi)genetic disorders in a heterogeneous cohort of men with reduced fertility.

Введение. Программное обеспечение, предоставляемое производителями автоматических генетических анализаторов, в большинстве случаев позволяет провести адекватный анализ результатов секвенирования ДНК по Сэнгеру для матриц с составом нуклеотидов, близким к эквивалентному. Однако для рассмотрения результатов секвенирования матриц, отличающихся неэквивалентным нуклеотидным составом, требуется проводить анализ электрофореграмм с сохранением информации об интенсивности сигналов флуоресценции. В особенности это касается секвенирования ДНК, модифицированной бисульфитом натрия. Цель: разработать и апробировать в практике научных исследований компьютерную программу для обеспечения адекватного анализа электрофореграмм секвенирования ДНК по Сэнгеру на основе бережного отношения к первичным данным и аккуратного определения базовых линий в спектральных каналах отдельных нуклеотидов. Методы. Программа SeqBase написана на языке C#, программная платформа .NET Framework 4.0, и выполняется в среде исполнения CLR (Common Language Runtime) для операционных систем семейства Windows. Адрес установочного пакета программы SeqBase: http://www.epigenetic.ru/projects/seqbase. Результаты. Разработана компьютерная программа, предназначенная для анализа первичных результатов секвенирования по Сэнгеру (хроматограмм капиллярного электрофореза), полученных на автоматических генетических анализаторах и представленных в файлах формата ABIF (*.ab1), обеспечивающая следующие возможности: 1) просмотр исходных электрофореграмм как в общем виде, так и раздельно по спектральным каналам; 2) кадрирование области анализа; 3) сглаживание сигналов; 4) ручная установка базовой линии по каждому из спектральных каналов; 5) сведение базовых линий по всем каналам; 6) ручная коррекция подвижности фрагментов ДНК в зависимости от типа флуоресцентной метки терминирующего нуклеотида. Апробация программы успешно проведена в рамках ряда исследований, результаты которых опубликованы в рецензируемых научных изданиях. Заключение. Использование программы SeqBase целесообразно для анализа результатов секвенирования по Сэнгеру матриц ДНК с неэквивалентным нуклеотидным составом, в особенности, модифицированных бисульфитом натрия, во избежание получения ложных результатов и для уточнения количественных оценок. Background. The software provided by the manufacturers of automatic genetic analyzers, in most cases, allows an adequate analysis of the results of Sanger DNA sequencing for templates with a nucleotide composition close to the equivalent. However, to consider the results of sequencing of templates with non-equivalent nucleotide composition, it is necessary to analyze electrophoregrams with preservation of primary information on the intensity of fluorescence signals. This is especially important for the sequencing of DNA modified with sodium bisulfite. Aim: to develop and validate in the practice of scientific research a computer program that ensures adequate analysis of electrophoregrams of Sanger DNA sequencing based on preservation of the primary data and on accurate determination of baselines in the spectral channels of individual nucleotides. Methods. The SeqBase program is written in C#, the programming platform .NET Framework 4.0, and runs in the CLR (Common Language Runtime) for Windows operating systems. SeqBase installation package address is http://www.epigenetic.ru/projects/seqbase. Results. A computer program has been developed designed to analyze the primary results of Sanger sequencing (chromatograms of capillary electrophoresis) obtained from automatic genetic analyzers and presented in files of the ABIF (*.ab1) format, which provides the following functions: 1) viewing the original electrophoregrams both in general form and separately by spectral channels; 2) cropping the area of analysis; 3) signal smoothing; 4) manual setting of the baseline for each of the spectral channels; 5) convergence of baselines on all channels; 6) manual correction of the mobility of DNA fragments depending on the type of fluorescent label of the terminating nucleotide. The program has been successfully tested in a number of studies, the results of which have been published in peer-reviewed scientific journals. Conclusion. The use of the SeqBase program is advisable for the analysis of the results of Sanger sequencing of DNA templates with non-equivalent nucleotide composition, especially those modified with sodium bisulfite, to avoid false results and to clarify quantitative estimates.

Для понимания особенностей регуляции и механизмов взаимодействия белков матрикса и трансмембранных рецепторов и их роли в поддержании структуры и функции ткани молочной железы в норме и при канцерогенезе важно установить вклад изменений экспрессии этих белков в процессы развития, роста, инвазии и метастазирования опухолей. Изучение статуса метилирования генов, кодирующих белки ламининов, нидогенов, интегринов, кадгеринов, матриксных металлопротеиназ и их ингибиторов в нормальной и опухолевой ткани необходимо для установления роли этих генов и механизмов их регуляции при раке молочной железы. Нами проведено исследование состояние метилирования этих генов в нормальных и опухолевых тканях молочной железы с целью поиска эпигенетических маркеров, ассоциированных с клинико-морфологическими характеристиками опухолей. To understand mechanisms of interaction between extracellular matrix proteins and transmembrane receptors, as well as their role in maintaining the structure and function of breast tissue in norm and in cancer, an important issue is to determine the contribution of changes in the expression of these proteins to the processes of development, growth, invasion and metastasis of breast cancer. Studies of the methylation status of genes encoding laminins, nidogens, integrins, cadherins, matrix metalloproteinases and their inhibitors in normal and tumor tissues are necessary to determine the roles of these genes and the mechanisms of their regulation in cancer. We have studied methylation status of these genes in normal and tumor tissues of the mammary gland in order to search for epigenetic markers associated with the clinical and morphological characteristics of the tumors.

To understand mechanisms of interaction between extracellular matrix proteins and transmembrane receptors, as well as their role in maintaining the structure and function of breast tissue in norm and in cancer, an important issue is to determine the contribution of changes in the expression of these proteins to the processes of development, growth, invasion and metastasis of breast cancer. Studies of the methylation status of genes encoding laminins, nidogens, integrins, cadherins, matrix metalloproteinases and their inhibitors in normal and tumor tissues are necessary to determine the roles of these genes and the mechanisms of their regulation in cancer. We have studied methylation status of these genes in normal and tumor tissues of the mammary gland in order to search for epigenetic markers associated with the clinical and morphological characteristics of the tumors.

AIM:microdeletions in the AZF region of Y-chromosome, compound heterozygotes of severe and mild CFTR mutations, and long CAG-repeats in the androgen receptor gene (AR) as marker of predisposition are frequently studied as genetic causes of male infertility. A simultaneously testing of the panel including biochemical, immunological, cyto- and molecular genetic markers is often performed during the complex laboratory diagnostics in infertile men. The aim of our work was to identify molecular genetic alterations, which are advisable for simultaneously testing in a man with currently uncertain form of infertility, to increase the informativeness of laboratory diagnostics.MATERIALS AND METHODS:a retrospective study of 885 infertile men was conducted. AZF deletions were determined by multiplex PCR using 10 STS-markers (sY83, sY84, sY86, sY127, sY134, sY143, sY152, sY157, sY254, sY255) and two control loci SRY and AMEL with detection in polyacrylamide gel. Mutations in the CFTR gene (F508del, CFTRdel2.3(21kb), I507del, 1677delTA, 2143delT, 2184insA, 394delTT, W1282X, G542X, N1303K, R334W and 5T) were detected by PCR and SNaPshot. For determination of length of the AR CAG-repeat a fragment analysis of fluorescently labeled PCR products on the 3500xl capillary sequencer was performed.RESULTS:AZF deletions were detected in 8.2% of cases. The largest number of deletions was found in the AZFc subregion (58.9%), while a frequency of deletion in AZFa, AZFb or combined deletions of two and three subregions was 5.5%, 12.3% and 23.3%, respectively. Heterozygous carriage of severe CFTR mutations was detected in 4.7% patients. The most frequent mutation was F508del (83.3%), followed by CFTRdel21kb (7.1%) and W1282X (4.8%). The frequency of the mild splicing 5T mutation was 5.3%, and its incidence was significantly higher than in the previously published control group (p=0.002). AR genotyping revealed that the most prevailing allele was 21 (CAG) (21.5%). Long alleles with 27 or more CAG-trinucleotides were identified in 7.5% of the tested cases. In addition, 7 CAG heterozygotes with Kleinfelter syndrome were found.CONCLUSION:during primary complex laboratory diagnostics in a heterogeneous group of infertile men, it is advisable to detect AZF deletions and the most frequent CFTR mutations, including F508del, CFTRdel21kb, 1677delTA, 2143delT, W1282X and 5T. The more comprehensive analysis of CFTR mutations is justified only in patients with verified obstructive infertility. Sequencing of panels associated with infertility genes using NGS technology is promising.

Background: Neoadjuvant chemotherapy (NAC) is intensively used for the treatment of locally advanced breast cancer (BC). The aim of this study is to provide a better insight into BC response to neoadjuvant anthracycline based chemotherapy. Methods: Genome-wide methylation analysis of 27 BC biopsy specimens of the luminal B subtype taken before the treatment was performed using the XmaI-RRBS method. Methylation status of the selected markers was next determined by methylation sensitive restriction enzyme PCR in a validating sample of 40 BC biopsy specimens. Results: By XmaI-RRBS we have identified 10 genes the states of methylation of which most effectively mark BC luminal B sensitivity to anthracycline based NAC. For locus specific assessment of these markers we have developed a multi-locus methylation sensitive PCR system. Based on the methylotyping results obtained for the 40 samples of the validating cohort, the diagnostic properties of the system were estimated: the area under the ROC curve was 84%, with the sensitivity of 82% and the specificity of 80%. Conclusions: The system including a limited number of methylation markers makes it possible to effectively predict the response of luminal B subtype BC to anthracycline based NAC by an analysis of biopsy material obtained before the treatment. Reasonable diagnostic sensitivity and specificity values are achieved only when the markers are evaluated in complex; in separate the differences in gene methylation frequencies between responding and non-responding tumors may be negligible (Table). Table80P Methylation frequencies of selected genes in tumors responding and not responding to anthracycline based NACGeneResponders, %Non-responders, %SLC9A328 (7/25)27 (4/15)C1QL220 (5/25)13 (2/15)DPYS60 (15/25)47 (7/15)IRF472 (18/25)33 (5/15)ADCY864 (16/25)40 (6/15)KCNQ248 (12/25)40 (6/15)TERT80 (20/25)60 (9/15)SYNDIG120 (5/25)20 (3/15)SKOR256 (14/25)67 (10/15)GRIK184 (21/25)87 (13/15) Open table in a new tab Legal entity responsible for the study: Research Centre for Medical Genetics. Funding: Russian Science Foundation (project No.18-15-00430). Disclosure: All authors have declared no conflicts of interest.

Matrix metalloproteinases and their inhibitors contribute to the regulation of cell-cell interactions and maintain tissue structure and function. We have analyzed DNA methylation at the promoter regions of 11 matrix metalloproteinases genes ( MMP2, MMP11, MMP14, MMP15, MMP16, MMP17, MMP21, MMP23B, MMP24, MMP25, MMP28 ), and 4 inhibitors of matrix metalloproteinases genes ( TIMP1, TIMP2, TIMP3, TIMP4 ). Collectively members of these families take part in regulation of matrix proteins degradation, signal transduction, adhesion, migration, cell differentiation, apoptosis and angiogenesis. Abnormal methylation frequencies at the promoter regions of genes MMP2, MMP23B, MMP24, MMP25, MMP28 in breast cancer accounted for 7,7%, 17%, 11,9%, 15,4% and 4,9% respectively. Promoter regions of genes TIMP1, TIMP4, MMP14 MMP21 were constitutively methylated in breast tissue.

Testicular cancer is the most common form of solid cancer in young men. Testicular cancer is represented by testicular germ cell tumors (TGCTs) derived from embryonic stem cells with different degrees of differentiation in about 95% of cases. The development of these tumors is related to the formation of a pool of male germ cells and gametogenesis. Clinical factors that are predisposed to the development of germ-cell tumors include cryptorchidism and testicular microlithiasis, as well as infertility associated with the gr/gr deletion within the AZFс locus. KITLG, SPRY4, and BAK1 genes affect the development of the testes and gametogenesis; mutations and polymorphisms of these genes lead to a significant increase in the risk of the TGCT development. To determine the relationship between gene polymorphisms and the development of TGCTs, we developed a system for detection and studied the allele and genotype frequencies of the KITLG (rs995030, rs1508595), SPRY4 (rs4624820, rs6897876), and BAK1 (rs210138) genes in fertile men, patients with TGCTs, and patients with infertility that have the AZFс deletion. A significant association of rs995030 of the KITLG gene with the development of TGCTs (p = 0.029 for the allele G, p = 0.0124 for the genotype GG) was revealed. Significant differences in the frequencies of the studied polymorphisms in patients with the AZFc deletion and the control group of fertile men were not found. We showed significant differences in the frequencies for the combination of all high-risk polymorphisms in the control group, patients with the AZFc deletion and patients with TGCTs (p (TGCTs-AZF-control) = 0.0207). A fivefold increase in the frequency of the combination of all genotypes in the TGCT group (p = 0.0116; OR = 5.25 [1.44‒19.15]) and 3.7-fold increase was identified in patients with the AZFc deletion (p = 0.045; OR = 3.69 [1.11‒12.29]) were revealed. The genotyping of patients with infertility caused by the AZFc deletion can be used to identify individuals with an increased risk of TGCTs.

Extracellular glycoproteins of the laminin family are essential components of basement membranes involved in a number of biological processes, including tissue differentiation, wound healing, and tumorigenesis. We present the first comprehensive study of promoter methylation status of the genes encoding laminin chains in normal tissues (peripheral blood leucocytes, buccal epithelial cells, autopsy breast tissue samples) and in breast carcinoma samples. Based on the results of this study, we divide laminin genes into three categories. Genes, constitutively methylated in breast tissues include LAMA3A, LAMB2, LAMB3, and LAMC2. Genes prone to abnormal methylation in breast carcinoma include LAMA1, LAMA2, LAMA3B, LAMA4, LAMB1, and LAMC3. Genes that are rarely if ever methylated in breast carcinoma include LAMA5 and LAMC1. The constitutively methylated group includes all of the genes that encode subunits of laminin-5 (the historical name of laminin 332), the promoters of which were previously considered unmethylated in normal tissues and prone to abnormal methylation in breast cancer.

The article discusses results of the structural and functional analysis of molecular genetic abnormalities in various malignant tumors. Investigations have discovered more than 20 new markers for sporadic breast cancer. Several of them formed the test system, allowing the diagnosis with a specificity of 100%. Appearance of TMPRSS2/ERG4 chimeric gene is a frequent tumor-specific event, its expression is correlated with more aggressive forms of prostate cancer, may serve as a molecular marker for tumor cells and androgen assessment of tumor response to hormonal therapy. The effective systems for the early diagnosis of cervix and endometrium cancer were developed as well. Mutations in the VHL, deletions of chromosome 3 and methylation of several genes can predict the course and selection of effective therapy of clear cell kidney cancer, a number of molecular markers were identified for early diagnosis and prognosis of recurrence of bladder cancer. For diagnosis, prognosis and treatment of brain tumors we developed an effective complex system of markers. Protocol of molecular genetics investigation reveals the cause of the disease by more than 90% of patients with retinoblastoma. In order to study abnormal methylation in tumor genomes an innovative technology AFLOAT has been developed that allows to efficiently identify new markers with diagnostic value. Test systems of molecular genetic and epigenetic markers for early diagnosis and prognosis as well as for cancer therapy optimization have shown to be effective, have been approved for use in clinical practice and are being introduced into practical healthcare.