Retinoblastoma (RB) is one of the most common malignant neoplasms of the visual organ in children, with an established genetic factor in the development of the disease. The detection of mutations in the RB1 gene has been successfully carried out for many years, however, the development of technologies for DNA diagnostics of RB allows us to consider this disease not only as an independent tumor, but also as one of the manifestations of more complicated genetic syndromes involving a complex of genes. The correct genetic diagnosis is important to understand the prognosis of the health of a patient with RB and recommendations for his further observation, as well as a correct assessment of the risks of having children with the same pathology in the family. Currently available methods of DNA diagnostics of the RB1 gene are new generation sequencing (NGS) and multiplex amplification of ligated probes (MLPA). However, in some cases, additional diagnostics may be required – a karyotype study or chromosomal micromatrix analysis (CMA). In this article we present a molecular genetic study of children with uni- and bilateral RB with an extended deletion at the RB1 gene locus. The aim of the study was determining of possible indications for the appointment of extended DNA diagnostics.

Introduction. Gastric cancer remains one of the most common cancers and has a high mortality rate worldwide. Epigenetic alternations of non-coding RNAs (ncRNAs), including microRNAs and long ncRNAs can contribute to its pathogenesis and progression, and could be potent diagnostic and prognostic biomarkers.Aim. Estimation of PROX1‑AS1 and miR-647 expression in gastric cancer and investigation of its clinical significance. Materials and methods. Tumor and adjacent normal tissues (n = 62), and sectional normal tissue samples (n = 5) were included in the study. The expression of the ncRNAs was quantified by reverse transcription-polymerase chain reaction assay.Results. We have reviled the significant difference in the PROX1‑AS1 expression in tumor (p = 0.002) and non-tumor tissues (p <0.001) obtained from gastric cancer patients in comparison with sectional gastric tissues without pathology. Pearson correlation analysis confirmed a negative correlation between PROX1‑AS1 and miR-647 in gastric cancer both in tumor (р <0,001) and adjacent normal tissues (р <0.001). Besides, expression of PROX1‑AS1 and miR-647 was associated with the size and extent of the primary tumor.Conclusion. The obtained results allow to suggest a potential prognostic value of PROX1‑AS1 and miR-647 in gastric cancer.

Epigenetic processes play a significant role in carcinogenesis, cancer recurrence and metastasis, and may serve as useful clinical biomarkers. Analysis of epigenetic regulator (ER) genes mutation landscape in gastric cancer (GC) samples may reveal novel genetic variants and therapeutic targets. This study presents an application of epigenetic regulators targeted NGS panel to GC patients. We investigated somatic mutations of 25 epigenetic regulation genes using the NGS panel in 95 gastric cancer samples. We revealed 1995 unique variants: 301 synonymous substitutions, 414 nonsynonymous substitutions, 34 nonsense mutations in 95 samples of gastric cancer. For further analysis, we selected frame shift mutations, nonsense and missense not annotated in the ClinVar, COSMIC, dbSNP databases and/or substitutions with MAF frequency < 0.0005. We studied survival the survival of patients using the Kaplan-Meier method. Based on these criteria, we identified 60 somatic mutations in 45/95 (48%) samples; in 50/95 (52%) samples, we did not find the corresponding mutations. All identified somatic variants were verified in tumor non-tumor tissue by Sanger sequencing. Somatic mutations are distributed as follows KMT2D -8, ARID1A-7, CHD7-6, KMT2A-6, KMT2C-5, CHD5-4, DNMT3B-3, MBD1-3, ARID2-2, CREBBP-2, SMARCA2-2, SMARCA4-2, HDAC2-2, CHD4-2, SIRT1-2, DNMT1- 1, TET1-1, EP300-1, EZH-1. All identified variants were investigated using the predictor programs PolyPhen2, PROVEAN, SNPs & GO, and MutPred2 and analyzed for pathogenicity. 28/60 mutations were identified as pathogenic, 9/28 (32%) were verified in non-neoplastic tissue, and were germline variants with a low frequency, or previously not described germline variants. 23/60 (38%) of the identified variants are new, not described in the databases. When assessing the survival of patients using the Kaplan-Meier method, we showed that survival was significantly reduced in the presence of somatic mutations in the epigenetic regulation genes and tumor size of T4, compared with the T4 group without mutations (p = 0.043), patients have mutations in the ER genes and distant metastases (M1), compared with the group without mutations (p < 0.0001). It was also revealed that mutations in ARID1A prevail in patients with distant metastases (p = 0.03). Although, of course, clinical associations are somewhat premature, since the group of patients is not very large yet.

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.

Almost 80% of cases of hereditary retinoblastoma do not have a family history and arise as a result of de novo mutations in the RB1 gene. An NGS test was performed on 208 unrelated patients with sporadic RB, including 145 patients with a unilateral form and 63 patients with a bilateral one. In the group of patients with bilateral RB, pathogenic variants in the RB1 gene were detected in 90.5% (57/63) cases. In 4.8% (3/63) of patients, a mosaic variants were determined. In the group of patients with unilateral RB, changes in the RB1 gene were detected in 17.9% (26/145) cases. Among the examined patients, somatic mosaicism was detected in 9.0% (13/165) cases. NGS allows us to determine the allelic frequency of variants, which makes the search for somatic mosaicism effective.

Проведено комплексное молекулярно-генетическое обследование пациентов с диагнозом туберозный склероз. Комплекс включает высокопроизводительное параллельное секвенирование с глубоким покрытием для выявления точковых мутаций и инделов, MLPA для выявления протяженных делеций и секвенирование по Сэнгеру. В выборке из 202 пациентов мутации найдены в 96,5% случаев; из них точковые мутации и инделы - в 93,3%, а протяженные делеции - в 6,7%. В 5,9% были найдены мутации с низкой аллельной представленностью (мозаицизм). A comprehensive molecular genetic examination of patients with diagnosis of tuberous sclerosis was carried out. The complex includes NGS with deep sequencing for detecting point mutations and indels, MLPA for identification of extended deletions, and Sanger sequencing. Mutations was found in 96,5% cases from 202 samples. Point mutation and indels were found in 93,3% cases, extended deletions were found in 6,7%. Mutations with low allelic representation were found in 5,9%.

Эпигенетические механизмы регулируют структуру хроматина и создают устойчивые закономерности экспрессии генов в процессе жизни клеток. Нарушение эпигенетической регуляции играет значительную роль в канцерогенезе, инвазии, рецидивировании и метастазировании опухолей и может служить полезным клиническим маркером. Мутационное профилирование генов эпигенетической регуляции в опухолевых образцах рака желудка позволит определить новые клинические и прогностические маркеры и дополнительные таргеты для лечения пациентов. В статье представлены первые результаты исследования соматических мутаций в генах эпигенетической регуляции, проведенного методом NGS. Epigenetic mechanisms regulate chromatin structure and create stable patterns of gene expression during cell life. Violation of epigenetic regulation plays a significant role in carcinogenesis, invasion, recurrence and metastasis of tumors, and can serve as a useful clinical marker. Mutational profiling of epigenetic regulation genes in tumor samples of gastric cancer will allow us to identify new clinical and prognostic markers and additional targets for the treatment of patients with gastric cancer. This article presents the first results of a study of somatic mutations in the epigenetic regulation genes carried out using NGS.

Background. Tuberculosis therapy can lead to considerably dissimilar outcomes among individual patients. Some patients benefit from anti-tuberculosis chemotherapy leading to a desired therapeutic effect, while other patients show insufficient or absent response, or develop adverse side effects. Individual response to isoniazid is affected by mutations in the gene encoding N-acetyltransferase 2 (NAT2). Wide variations were observed in distributions of polymorphic NAT2 alleles in human populations depending on race, ethnicity, or geographical origin. No data are available to date on prevalence of NAT2 gene polymorphic variants and acetylation types in Yakut (Sakha) patients with newly diagnosed pulmonary tuberculosis. Aims: the frequencies of NAT2 polymorphic SNPs as well isoniazid acetylation phenotypes were evaluated in the patients cohort of Yakut ethnicity with newly diagnosed pulmonary tuberculosis (PTB). The comparison with, other ethnic groups of Asian origin was done. Methods. Cross-sectional study was conducted among Yakut patient cohorts with newly identified pulmonary tuberculosis. Using real-time PCR the following SNPs were explored: NAT2*5 (rs1801280, Т341С), NAT2*6 (rs1799930, G590A), NAT2*7 (rs1799931, G857A), NAT2*11 (rs1799929, C481T), NAT2*12 (rs1208, A803G), NAT2*13 (rs1041983, C282T). Genetically determined metabolic rates were calculated using NATpred online tool. Results. NAT2 SNPs were assessed, namely *5, *6, *7, *11, *12, and *13. The most frequent in Yakut patients were NAT2*6 and NAT2*13 SNPs (40.9% and 64.4%, respectively). Significant differences were detected in frequencies of NAT2 *5, *11, *12 polymorphisms; all studied NAT2 gene polymorphisms showed meaningful differences in genotype and minor allele prevalence rates after comparison between Yakut population and other Mongoloid ethnic groups (populations of China, Japan, Vietnam). High prevalence of intermediate acetylation type among ethnic Yakuts (58.3%) was established. Conclusions. Certain distinct differences in allelic variants of NAT2 gene and acetylation type prevalence in patients of Yakut ethnic origin newly diagnosed with pulmonary tuberculosis let conclude that the existing genotyping and phenotyping data from studies among other Mongoloid populations cannot be implicitly extrapolated on Yakut people. Pharmacogenetic data on individual response to drugs in Yakut patients should be made use of in clinical practice, to develop personalized isoniazid administration algorithms, with a final goal to make treatment more effective and safe.

Epigenetic processes play a significant role in carcinogenesis, cancer recurrence and metastasis, and may serve as useful clinical biomarkers. According to GLOBOCAN 2018 data, gastric cancer (GC) is the third most lethal cancer with about 783000 deaths in 2018. Therapeutic drugs that are effective in the treatment of various types of tumors have a weak therapeutic effect in the treatment of GC due to the lack of genetic variants in known driver genes. Analysis of epigenetic regulator genes mutation landscape in GC samples may reveal novel genetic variants and therapeutic targets. This study presents an application of epigenetic regulators targeted NGS panel to GC patients. We designed the NGS-based targeted panel of 25 genes whose products are involved in epigenetic processes and determined somatic alterations in 52 tumor samples of the GC. This panel consists of genes that regulate DNA methylation: DNMT1, MBD1, TET1, DNMT3A, DNMT3B ; genes involved in the modification of histone proteins: EZH2, UTX, EP300, JARID1B, CREBBP, HDAC2, SIRT1, KMT2A, KMT2D, KMT2C ; chromatin remodeling genes: SMARCB1, SMARCA2, SMARCA4, ARID1A, ARID2, BRD7, PBRM1, CHD5, CHD7, CHD4 . For the selection of genes, we took into account the frequency of somatic variants in GC according to the COSMIC cancer database. Prediction of somatic variants pathogenicity and impact on structure were carried out using PolyPhen2, SIFT, PROVEAN, MutPred2, I-Mutant 3.0 and HOPE3D tools. All identified somatic variants were verified in tumor tissue by Sanger sequencing. In 52 GC samples, targeted NGS sequencing revealed 50 nonsynonymous substitutions, 20 frameshift indels, 5 nonsense variants. We selected only known variants with minor allele frequency (MAF) lower than 0.0001 and no available information about clinical significance or novel genetic variants, which have never been described in any database. Of these selected variants, 11 are known with low MAF, and 14 are novel. Based on results of in silico pathogenicity prediction, substitutions in genes ARID1A (p.R2236C, p.Q152*, p.S1828*), KMT2A (p.W1909*), KMT2D (p.D3419G), KMT2C (p.Q462*, p.P959I, p.R973G), SMARCA4 (p.P913L) and CHD4 (p.R1943Q) are of interest due to pathogenicity prediction and disruption of various molecular mechanisms such as ligand binding, molecular recognition and relative solvent accessibility. In this study, we present novel results on somatic mutation profiling in epigenetic regulators for GC patients. The identified pathogenic variants can be used as prognostic markers or new drug targets, but further investigation is needed. Deep target sequencing of epigenetic regulators makes it possible to acquire novel alterations and resolve true mutation frequencies of such genes. Subsequently, results of mutational landscape studies can be used to form risk groups among patient, confer significant prognostic information and improve clinical decision-making.

Epigenetic mechanisms regulate chromatin structure and create stable patterns of gene expression during cell life. Violation of epigenetic regulation plays a significant role in carcinogenesis, invasion, recurrence and metastasis of tumors, and can serve as a useful clinical marker. Mutational profiling of epigenetic regulation genes in tumor samples of gastric cancer will allow us to identify new clinical and prognostic markers and additional targets for the treatment of patients with gastric cancer. This article presents the first results of a study of somatic mutations in the epigenetic regulation genes carried out using NGS.

Почти 80% случаев наследственной ретинобластомы не имеют семейного анамнеза и возникают в результате мутаций de novo в гене RB1. Методом высокопроизводительного параллельного секвенирования (ВПС) проведено молекулярно-генетическое обследование 208 неродственных больных со спорадической РБ, среди которых 145 пациентов с унилатеральной формой заболевания и 63 - с билатеральной. В группе пациентов с билатеральной РБ молекулярные изменения в гене RB1 обнаружены в 90,5% (57/63) случаев. У 4,8% (3/63) пациентов определен мозаичный вариант мутации в гене RB1. В группе пациентов с унилатеральной РБ молекулярные изменения в гене RB1 выявлены в 17,9% (26/145) случаев. Среди исследованных пациентов соматический мозаицизм выявлен в 9,0% (13/165) случаев. Применение ВПС позволяет точно определять аллельную частоту вариантов, что делает поиск соматического мозаицизма эффективным. Almost 80% of cases of hereditary retinoblastoma do not have a family history and arise as a result of de novo mutations in the RB1 gene. An NGS test was performed on 208 unrelated patients with sporadic RB, including 145 patients with a unilateral form and 63 patients with a bilateral one. In the group of patients with bilateral RB, pathogenic variants in the RB1 gene were detected in 90.5% (57/63) cases. In 4.8% (3/63) of patients, a mosaic variants were determined. In the group of patients with unilateral RB, changes in the RB1 gene were detected in 17.9% (26/145) cases. Among the examined patients, somatic mosaicism was detected in 9.0% (13/165) cases. NGS allows us to determine the allelic frequency of variants, which makes the search for somatic mosaicism effective.

A comprehensive molecular genetic examination of patients with diagnosis of tuberous sclerosis was carried out. The complex includes NGS with deep sequencing for detecting point mutations and indels, MLPA for identification of extended deletions, and Sanger sequencing. Mutations was found in 96,5% cases from 202 samples. Point mutation and indels were found in 93,3% cases, extended deletions were found in 6,7%. Mutations with low allelic representation were found in 5,9%.

Целью исследования было охарактеризовать клинико-генетические корреляции у пациентов с ABCA4-ассоциированными наследственными заболеваниями сетчатки (НЗС). Распространенная «мягкая» мутация АВСА4:p.G1961E была обнаружена у 20 из 54 больных, наблюдающихся с НЗС и имеющих, по крайней мере, одну мутацию в гене АВСА4. У 8 пациентов p.G1961E выявлена в компаунд-гетерозиготном состоянии с комплексной миссенс-мутацией p.[L541P;A1038V], у 6 - с другими миссенс-мутациями (p.N1805D; p.R1640W; p.P1088T; p.L541P; p.P1380L; p.R1640Q). В нашей выборке у пациентов с длительностью заболевания от 9 лет мутация p.G1961E в 81% случаев ассоциирована с лёгким течением НЗС, даже при наличии второй тяжелой мутации, и является предиктором более легкого течения заболевания, в соответствии с базовой моделью клинико-генетических корреляций при АВСА4-ассоциированных заболеваниях. The aim of this study was to characterize phenotype-genotype correlations in patients with ABCA4-associated IRDs. By NGS, we have identified the widespread “mild” ABCA4 mutation p.G1961E in 20 of 54 IRD patients with at least one mutation in the ABCA4 gene. In 8 patients, p.G1961E was in combination with a complex missense mutation p. [L541P; A1038V], in six - in combination with other missense mutations (p.N1805D; p.R1640W; p. P1088T; p.L541P; p.P1380L; p.R1640Q). In 81% of our patients with a disease course of 9 years or more, the p.G1961E mutation is associated with mild disease, even in the presence of a second severe mutation, and is a predictor of a milder phenotype, in accordance with the basic model of phenotype-genotype correlations in ABCA4-associated diseases.

Актуальность. При наследственной ретинобластоме герминальная мутация в одном из аллелей гена RB1 обусловливает предрасположенность к заболеванию и его семейную передачу. Наследственная ретинобластома манифестирует в более раннем возрасте по сравнению со спорадической формой и носит в большинстве случаев мультифокальный и билатеральный характер. Однако некоторые семьи с ретинобластомой (два и более носителя одинаковой герминальной мутации в родословной) демонстрируют более мягкий фенотип с неполной пенетрантностью (у части носителей герминальной мутации заболевание не развивается) и вариабельной экспрессивностью (одинаковая мутация у разных членов семьи может проявляться уни- или билатеральной формой заболевания). Выявление низкопенетрантных мутаций в гене RB1 и изучение характера их наследования способствует пониманию механизмов, лежащих в основе развития наследственной ретинобластомы с низкой пенетрантностью, и крайне важно как для дальнейшего расширения знаний о молекулярной генетике ретинобластомы, так и для медико-генетического консультирования и последующего клинического ведения семей с такой формой заболевания. Цель. Установить спектр генетических нарушений в гене RB1 у больных с наследственной ретинобластомой с неполной пенетрантностью и вариабельной экспрессивностью в выборке российских пациентов и определить влияние родительского происхождения мутации в RB1 на её фенотипическое проявление. Методы. Методом высокопроизводительного параллельного секвенирования проведено молекулярно-генетическое обследование 332 неродственных больных с ретинобластомой. Для верификации выявленных точковых мутаций и анализа их сегрегации в родословных использовали секвенирование по Сэнгеру. Результаты. В группе пациентов без семейного анамнеза ретинобластомы у 3,5% больных определен наследственный характер заболевания, при котором один из родителей являлся бессимптомным носителем герминальной мутации в гене RB1. Выявлено 10 низкопенетрантных мутаций в гене RB1 и установлен спектр мутаций, приводящих к развитию наследственной ретинобластомы с низкой пенетрантностью. В 91,7% случаев наследственной ретинобластомы с низкой пенетрантностью мутантный аллель получен пробандом от отца, у которого отсутствовали клинические признаки заболевания или наблюдалась более легкая форма. Выводы. Полученные результаты подтверждают ранее высказанные предположения, что низкопенетрантные герминальные мутации в гене RB1, унаследованные от отца, чаще приводят к развитию ретинобластомы, и в более тяжелой форме по сравнению с таковыми, унаследованными от матери. Background. In hereditary retinoblastoma, a germline mutation in one of the alleles of the RB1 gene causes a predisposition to the disease and its transmission within the pedigree. Hereditary retinoblastoma manifests at an earlier age compared with the sporadic form and in most cases is multifocal and bilateral. However, some families with retinoblastoma (two or more carriers of the same germline mutation in the pedigree) exhibit a milder phenotype with incomplete penetrance (some carriers of the germline mutation are asymptomatic) and variable expressivity (the same mutation in different family members may manifest as either a unilateral or bilateral form). Identification of low-penetrant mutations in the RB1 gene and studying their inheritance in pedigrees contributes to understanding the mechanisms underlying the development of retinoblastoma with low penetrance. It is extremely important both for further expansion of knowledge in the field of molecular genetics of retinoblastoma, and for competent genetic counseling and subsequent clinical management of families with this form of the disease. Objective. To establish the spectrum of genetic disorders in the RB1 gene in Russian patients with hereditary retinoblastoma with incomplete penetrance and variable expressivity and to determine the effect of the parental origin of the RB1 mutation on its phenotypic manifestation. Methods. Using high-performance parallel sequencing, a molecular genetic survey of 332 unrelated patients with retinoblastoma was performed. Sanger sequencing was used to verify the identified point mutations and analyze their segregation in pedigrees. Results. In the group of patients without a family history of retinoblastoma, in 3.5% the hereditary nature of the disease was determined, where one of the parents was an asymptomatic carrier of a germline mutation in the RB1 gene. Ten low-penetrant mutations in the RB1 gene were identified. In 91.7% of cases of hereditary retinoblastoma with low penetrance, the mutant allele was obtained by a proband from a father with no clinical signs of the disease or with a milder form. Conclusion. The results confirm the previously suggested assumptions that low-penetrant germline mutations in the RB1 gene inherited from the father more often lead to the development of retinoblastoma, and in a more severe form than those inherited from the mother.

Mycosis fungoides (MF) is the most common primary cutaneous epidermotropic T-cell lymphoma (80%). The accurate diagnosis of MF confirmed only by clinical, histological and immunohistochemical signs amounts to 50-75%.OBJECTIVE:To investigate genetic markers (FOXP3, STAT4, IL-12B) for the early diagnosis of MF, to estimate the informative value of used diagnostic techniques (histology, immunophenotyping), and to determine clonality by the T-cell receptor γ-chain genes.MATERIAL AND METHODS:Fifty patients with MF and plaque parapsoriasis (PP) who had been treated at the V.A. Rakhmanov Clinic of Skin and Venereal Diseases and at the National Medical Research Center for Hematology were followed up. A MF group consisted of 27 patients; a PP group included 23 patients, and a control group comprised 10 healthy individuals. The expression of the FOXP3, STAT4, and IL-12B genes was analyzed by TaqMan real time-PCR. The objectives of the study were affected skin portions from patients with MF or PP and healthy individuals.RESULTS:The investigation revealed a increase in the expression level of STAT4 mRNA transcripts by 9 times in patients with MF compared with those with PP and by 553 times in healthy individuals. There was also a statistically significant predominance of the expression level of STAT4 mRNA transcripts in patients with spotted and plaque stages of MF (180; 318) compared with those with PP and healthy individuals, as well as a sharp decrease in those with erythrodermic MF, which was statistically significant.CONCLUSION:MF cannot be diagnosed without comprehensively assessing the clinical, anamnestic, histological, immunophenotypic, and molecular genetic data. The expression level of STAT4 mRNA transcripts is of great importance for the early diagnosis of MF. Inclusion of the level of STAT4 expression in the list of diagnostic signs increases the accuracy of differential diagnosis of MF and PP from 59.1 to 81.8%, respectively.

Background Gastric cancer is the fifth most common malignancy in the world and the third leading cause of cancer death. Approximately 1–3% of gastric cancers are hereditary. Mutations of CDH1,the gene encoding E-cadherin, are the most common germline mutations detected in gastric cancer and cause hereditary diffuse gastric cancer (HDGC) syndrome. However, there has been no research previously conducted to identify candidate genes for predisposition to hereditary gastric cancer in the Russian population. The purpose of our research was to fill this gap. Methods For this study, we collected specimens of the venous blood of 40 patients (age up to 50 years) diagnosed with diffuse gastric cancer, and 80 gastric cancer tumor samples. To define the mutation in these patients we have used NGS with two panels -an Ion AmpliSeq Cancer Hot spot Panel v2, widely used for the analysis of «hot spots» in cancer related genes, and an in-house panel developed for sequencing the genes involved in gastric carcinogenesis (BMPR1A, SMAD4, CDH1, TP53, STK11, PTEN). All found mutations were verified in DNA obtained from peripheral blood lymphocytes, using polymerase chain reaction followed by Sanger sequencing. Results In total, with two panels, we have identified 16 genetic germline variants with exceptionally low general population frequencies (no higher than 0.00004 according to the gnomAD database), and 4 genetic variants that have never been reported previously. We detected 6 deleterious mutations in CDH1 gene. All found mutations were missense mutations (A2512G:p.S838G, c.G1234A:p.V412I, .G2635A:p.G879S, c.C8G:p.P3R, c.C670T:p.R224C). Among these 6 mutations, one (c.A907c:pT303P) was newly discovered in this study. Other deleterious germline variants were found in APC, CDKN2B, STK11, MET, SMO and two in RB1 genes. The mean age at diagnosis of gastric cancer patients with mutation was 46,1. Conclusions Identifying mutation carriers in families is extremely important. The identification of germline CDH1 mutations offers the opportunity for potentially lifesaving prophylactic gastrectomy, which is the standard of care for these individuals. Legal entity responsible for the study The authors. Funding Russian Foundation for Basic Research (project No. 18-015-00333), Russian Foundation for Basic Research (project No. 18-29-09020). Disclosure All authors have declared no conflicts of interest.

Introduction: This study was aimed to identify somatic and germline variants in Russian patients with gastric cancer (GC) by next-generation sequencing (NGS). Knowledge of the GC mutation spectrum helps to better understand its molecular pathogenesis and to evaluate the potential for targeted drug therapy. Methods: We recruited 52 patients, 34 males and 18 females, with locally advanced GC. For all patients gastrectomy was performed and tumor samples were investigated. To evaluate GC mutation profile, we conducted NGS for 52 tumor samples using the Ion AmpliSeq Cancer HotSpot panel (CHP) and our custom 6-gene (BMPR1A, SMAD4, CDH1, TP53, STK11, PTEN) hereditary GC (HGC) panel. Variant alleles were further assessed by Sanger sequencing in the adjacent non-tumor tissues in order to clarify their somatic or germline origin. Pathogenicity of genetic variants was evaluated with referral to gnomAD, ClinVar archive, and prediction of pathogenicity for novel variants was obtained using SIFT, PolyPhen2, SNPs&GO, Project HOPE 3D and MutationTaster software. Results: In 52 GC samples, NGS revealed 72 variants in 28 genes known to harbor mutation hotspots in cancer. CHP detected 39 variants in 22 genes, and 33 variants in 6 genes were found with our HGC panel. Of these, 63 genetic variants are known, and the remaining 9 are novel. Of 9 novel variants, one ( ATM :p.Glu3022=) is synonymous, 1 is frameshift ( STK11 :p.Ser283fs), 1 affects splicing ( CDH1 :c.1320 + 2T>G), 1 is nonsense ( SMO : p.Trp206*) and the remaining 5 ( SMAD4 :p.Val158Ala, STK11 :p.Met289Lys, CDH1 :p.Thr303Pro, RB1 :p.Leu564Phr, RB1 :p.His686Asn) are missense. Notably, 3 of the novel variants ( RB1 :p.Leu564Phr, CDH1 :p.Thr303Pro, and SMO : p.Trp206*) are germline and are predicted to be damaging and disease-causing by bioinformatic tools. Conclusion: Germline genetic variants obtained in this study are of undoubted interest for the diagnostics of hereditary GC. Pathogenic variants can have a direct relationship to the development of hereditary forms of GC and may lead to increased risk of relapse. Somatic genetic variants profiling will help to identify GC driver mutations. Novel genetic variants may serve as new targets for future therapies and help in understanding the pathobiology of GC. This work was supported by the Russian Foundation for Basic Research (project No. 18-015-00333), Russian Foundation for Basic Research (project No. 18-29-09020).

Background Retinoblastoma (RB) is the most common pediatric intraocular neoplasm caused by the biallelic inactivation of the RB1 tumor suppressor gene. In 40% of cases, the development of RB is mediated by a germline mutation in one of the alleles of RB1. Patients with germline mutation develop bilateral tumor with penetrance of more than 90%. However, some families demonstrate cases of RB with low penetrance (unaffected carries) and variable expressivity (carries develop either bi- or uni- RB). It is believed that the phenotypic manifestation of hereditary retinoblastoma depends on the functional type of the germinal mutation in the RB1. The molecular mechanisms underlying the variable phenotypic manifestation of the same mutation in different family members are currently explained by the parent-of-origin effect of RB1 mutation. Methods Using NGS of the RB1 we have analyzed DNA from blood of 331 unrelated patients with RB (226 patients with uni- RB and 105 with bi- RB). DNA samples of available family members were also examined for the presence of an identified mutation using Sanger sequencing. Results We identified 11 germline mutations in the RB1 that led to the RB with low penetrance and/or variable expressivity in 12 families. Among the identified mutations:, 25.0% - are missense mutations, 58.3% - are splice mutations and 16.7% - are frame shift mutations. In 91,7% of cases, probands inherited the mutant allele from their fathers, who were either clinically healthy carriers (7 families) or had the uni-/bilateral form of RB (3 families). Table: 1170P . Mutations Carrier (Proband, P) Form (uni, U; bi-lateral, B; no symptoms, -) c.1364G>C; c.1573G>A; c.1981C>T; с.607 + 1G-T (2 families); c.45_76del; с.83del P ♂ U - c.861G>C P ♀ U - c.939G>A P ♂ Grandfather Uncle U - - U c.380 + 1G-A; с.1695 + 5G-T; c.1696-2A-G P ♂ B U Conclusions The identification of mutations in the RB1 leading to the development of RB with low penetrance and variable expressivity, is necessary for adequate treatment and competent determination of the risk of developing the disease in other family members. Legal entity responsible for the study State assignment of Ministry of Science and Higher Education of the Russian Federation. Funding The state assignment of Ministry of Science and Higher Education of the Russian Federation. Disclosure All authors have declared no conflicts of interest.