Введение. Среди поясно-конечностных мышечных дистрофий (ПКМД) по частоте встречаемости дисферлинопатия занимает второе место в мире после кальпаинопатии. Заболевание характеризуется относительно поздней манифестацией, а сходная клиническая картина в группе ПКМД в ряде случаев создает значимые сложности в дифференциальной диагностике. Методом секвенирования нового поколения (NGS) можно быстро и эффективно определить вариант нуклеотидной последовательности гена DYSF, приводящий к нарушению синтеза белка дисферлина. Цель исследования: определить эффективность выявления вариантов нуклеотидных последовательностей в гене DYSF методом NGS у пациентов с клиническим диагнозом диферлинопатия и оценить возможности данного метода в дифференциальной диагностике ПКМД. Методы. Поиск генетических вариантов в гене DYSF производился у 157 пациентов при наличии клинических проявлений ПКМД, сопровождающихся повышением уровня креатинфосфокиназы (КФК) в 10-100 раз, манифестацией в возрасте 2-73 лет. У 27 пациентов выявлены изменения в последовательности гена DYSF. Методом Сэнгера было обследовано 9 из 27 человек – 34 (15-58)%. 18 пациентов было обследовано методом NGS, что составляет 67(42-85)%. У 2 родственных пациентов, у которых был выявлен только один вариант измененной последовательности гена DYSF, для поиска крупных делеций и (или) дупликаций была проведена мультиплексная лигаза-зависимая амплификация (MLPA). Выявленные методом NGS генетические варианты верифицировались референтным методом – ПЦР. Гистологическое и иммуногистологическое исследование выполнено четырем пациентам с неоднозначным результатом молекулярно-генетической диагностики. В качестве биоптата забран фрагмент (5 мм3 ) латеральной головки четырехглавой мышцы бедра. Результаты и выводы. Методом NGS в гене DYSF был выявлен 21 различный генетический вариант у 27 пациентов. 16 из 21 генетических вариантов ранее описаны; в четверти случаев (5 вариантов, 25(0-52)%) генетические варианты являются ранее не описанными (novel). Из 21 выявленного варианта на основании критериев ACMG 12 (57(31-81)%) были классифицированы как патогенные, 5 (25(0-51)%) – как вероятно патогенные, 4 (20(0-46)%) – как варианты с неизвестной клинической значимостью. Background. According to the frequency of occurrences dysferlinopathy occupies the second place in world among limb–girdle muscular dystrophy (LGMD) after calpainopathy. The disease has a relatively late manifestation and the similar clinical picture with other LGMD. In some cases, that creates significant difficulties in differential diagnostics. New generation sequencing (NGS) is method that quickly and efficiently allows to determine the variant of the DYSF gene that leads to violation of protein synthesis. Aim: to determine the effectiveness of identifying variants of nucleotide sequences in the DYSF gene by the NGS in patients with a clinical diagnosis of diferlinopathy and also to evaluate the possibilities of this method in differential diagnostics of LGMD. Methods. The search for genetic variants of the DYSF gene was performed in patients with detected phenotype of LGMD, as well as 10 to 100 times higher levels of creatine kinase (CK) and manifestation at the age of 2-73 years. In total, 157 patients with a clinical signs of LGMD were included in the study. Changes in the sequence of the DYSF gene were detected in 27 of them. 9 of 27 people – 34(15-58)% were examined with Sanger sequencing. 18 of them – 67(42-85)% were examined by the NGS method. In two patients only one changed variant in the DYSF gene was detected, in this regard multiplex ligation-dependent probe amplification (MLPA) was performed to search for large deletions and (or) duplications. The identified genetic variants were verified by the reference method – PCR. Histological and immunohistological examination was performed in four patients with ambiguous results. As a biopsy material for examination were used fragments (5 mm3 ) of the lateral head of the quadriceps femoral muscle. Results and conclusions. Using the NGS method, in 27 patients 21 genetic variant of the DYSF gene was identified. 16 variants out of 21 belong to the category of previously described; thus, in almost a quarter of cases (5) – 25(0-52)% variants weren’t described before (novel). Among the 21 identified variants, based on ACMG criteria, 12 – 57(31-81)% variants were classified as pathogenic, 5 – 25(0-51)% as probably pathogenic and 4 – 20(0-46)% as variants with uncertain significance, however, all detected variants were accompanied by a detailed clinical signs of LGMD.

Количество публикаций с описанием первично-множественных опухолей растет, тем не менее сообщения о синхронно-метахронных неоплазмах встречаются крайне редко. Более того, генетический анализ таких опухолей и возможных путей их прогрессии в литературе практически отсутствует. Целью данного исследования было установление независимости происхождения множественных опухолей у пациентки с синдромом Линча. Ретроспективный анализ материала предусматривал как изучение морфологических особенностей опухолей, так и выявление мутационных повреждений в исследуемых генах путем полноэкзомного секвенирования ДНК, выделенной из имеющихся в наличии парафиновых блоков. Совокупность гистологических типов и установленных мутационных профилей неоплазм подтверждает их независимое происхождение. Таким образом, в статье представлен новый случай первично-множественных опухолей и предположены возможные пути их формирования с участием врожденного нарушения системы коррекции неспаренных оснований. The number of cases with the description of primary multiple tumors is growing, however, reports of synchronous-metachronous neoplasms are extremely rare. Moreover, there is practically no genetic analysis of such tumors and possible ways of their progression in the literature. The purpose of this study was to establish the independence of the origin of multiple tumors in a patient with Lynch syndrome. A retrospective analysis of the material included both the study of the morphological features of tumors and the detection of mutational damage in the studied genes by whole exome sequencing of DNA isolated from available paraffin blocks. The combination of histological types and established mutational profiles of neoplasms confirms their independent origin. Thus, a new case of primary multiple tumors is presented and possible ways of their formation with the participation of congenital disorders of the DNA mismatch repair system are suggested.

Differential diagnosis of inflammatory myopathies with hereditary muscular dystrophies accompanied by a secondary inflammatory process is a time‑consuming clinical and pathomorphological task. In particular, false diagnosis of polymyositis in patients with dysferlinopathy reaches 25 % of cases.A 40‑year‑old female patient with a limb‑girdle phenotype of dysferlinopathy, initially diagnosed as polymyositis, is presented. The reasons that led to the erroneous diagnosis were: sporadic case; subacute onset; proximal muscle weakness; myalgia, which stopped on the glucocorticosteroid therapy; high levels of creatine phosphokinase (up to 17 times); the presence of lymphocytic‑macrophage infiltrate in the muscle biopsy and the absence of magnetic resonance imaging data in primary examination of the patient.The refractoriness of clinical and laboratory signs to complex immunosuppressive therapy was the reason for revising the muscle biopsy with typing of the inflammatory infiltrate. The predominantly unexpressed perivascular infiltrate was characterized by the predominance of macrophages and, to a lesser extent, CD4+, which indicated the secondary nature of the inflammation in the muscle observed in some hereditary muscular dystrophies. When conducting an immunohistochemical reaction, the absence of the dysferlin protein in the sarcoplasmic membrane was revealed.Whole‑exome sequencing (NGS) revealed a mutation in exon 39 of the DYSF gene (p.Gln1428Ter) in the heterozygous state, which leads to the appearance of a stop codon and premature termination of protein translation. MLPA method registered 3 copies of exons 18, 19, 20, 22, 24 of the DYSF gene.Thus, this clinical example reflects the main methodological errors and possible effects of immunosuppressive therapy in patients with dysferlinopathy.

The frequency of a genetic variant in a population is crucially important for accurate interpretation of known and novel variant effects in medical genetics. Recently, several large allele frequency databases, such as Genome Aggregation Database (gnomAD), have been created to serve as a global reference for such studies. However, frequencies of many rare alleles vary dramatically between populations, and population-specific allele frequency can be more informative than the global one. Many countries and regions (including Russia) remain poorly studied from the genetic perspective. Here, we report the first successful attempt to integrate genetic information between major medical genetic laboratories in Russia. We construct an expanded reference set of genetic variants by analyzing 6,096 exome samples collected in two major Russian cities of Moscow and St. Petersburg. An approximately tenfold increase in sample size compared to previous studies allowed us to identify genetically distinct clusters of individuals within an admixed population of Russia. We show that up to 18 known pathogenic variants are overrepresented in Russia compared to other European countries. We also identify several dozen high-impact variants that are present in healthy donors despite either being annotated as pathogenic in ClinVar or falling within genes associated with autosomal dominant disorders. The constructed database of genetic variant frequencies in Russia has been made available to the medical genetics community through a variant browser available at http://ruseq.ru.

Early epileptic encephalopathy-66 was first diagnosed in a male patient from Russia using whole-exome sequencing. Early epileptic encephalopathy- 66 is a unique disorder in the group of early epileptic encephalopathies. The same recurrent heterozygous variant of the nucleotide sequence was found in all known patients, but the severity of seizures and dysmorphic signs significantly vary between patients. The current study of a recurrent pathogenic variant in PACS2 gene expands the phenotype spectrum of early epileptic encephalopathy-66 and will improve the management of patients with that disorder in Russia in the future.

Изучение спектра мутаций и совершенствование диагностики синдрома Ашера (СА) особо актуальны в связи с разрабатываемыми подходами к генной терапии заболевания. Среди 46 пациентов с признаками СА патогенные мутации выявлены нами у 40 (87%) пациентов. СА I и II типов определены у 26% и 57% пробандов исходной выборки, соответственно. У пациентов с СА I выявлены мутации в генах MYO7A (73%), CDH23 (7%), PCDH15 (7%), и USH1C (13%). Наибольшую частоту показала мутация MYO7A p.Q18*. Описано 6 новых мутаций в гене MYO7A, и две - в гене PCDH15. У пациентов с СА II выявлена 21 мутация гена USH2A, 5 из которых описаны впервые. Наибольшую частоту показала мутация USH2A p.W3955*. У двух пациентов выявлены мутации в генах несиндромального пигментного ретинита RHO и RPGR, что позволило уточнить клинический диагноз. Studying the mutation spectrum and improvement of molecular verification of the Usher syndrome (USH) are of particular relevance as gene therapy emerges. Among 46 patients with signs of Usher syndrome we identified mutations in 40 (85%) patients, establishing a diagnosis of USH1 and USH2 for 26% and 57% of the probands of the initial sample, respectively. Patients with USH1 showed mutations in the MYO7A (73%), CDH23 (7%), PCDH15 (7%), and USH1C (13%) genes. MYO7A p.Q18* mutation showed the highest frequency. We have identified 6 new mutations in the MYO7A gene, and 2 in the PCDH15 gene. In USH2 patients, 21 USH2A gene mutations were identified, 5 of which are novel. The USH2A mutation p.W3955* was most frequent. Two patients showed mutations in the non-syndromic retinitis pigmentosa genes RHO and RPGR, which made it possible to clarify the clinical diagnosis.