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The MinION system (Oxford Nanopore Technologies, United Kingdom) was used to directly sequence genomic DNAs of two recombinant Escherichia coli strains. One strain carried a plasmid with the gene for M.HpaII DNA methyltransferase, which methylates the second cytosine in a CCGG site. The other strain contained M.HspAI DNA methyltransferase, which modifies the central cytosine in a GCGC sequence. Either enzyme methylates cytosine to produce 5-methylcytosine. The presence of 5-methylcytosine was found to be detectable with high accuracy when DNA is sequenced at high coverage. In particular, 98.9% of the GCGC sites were identified as methylated at the first cytosine when DNA from the strain carrying the cloned M.HspAI gene was sequenced at 1300× coverage. Only 0.09% of the remaining tetranucleotides with a central CpG dinucleotide were false positives and were identified as methylated at the central cytosine. For the strain with M.HpaII, 91.3% of all CCGG sites were identified as methylated in a set of positions covered by more than 700 reads and only 0.13% of the other tetranucleotides with a central CpG dinucleotide were identified as sites with 5-methylcytosine in the second position. Thus, coverage of at least 700–1000× is necessary for accurately measuring 5-methylcytosine and eliminating false-positive results when using the method.

Congenital core myopathies are a clinically and genetically heterogenous group of congenital myopathies that share a specific histopathological feature: areas of reduced oxidative activity in muscle fibers. The relationship between clinical, genetic and morphological characteristics of this group of disorders remains understudied. The aim of this work was to compare clinical presentations and morphological phenotypes of patients with congenital myopathies/myodystrophy to the data yielded by massively parallel exome sequencing. Eight children were included in the study: 2 boys and 6 girls aged 3 to 14 years. Their biopsy material was analyzed by light and electron microscopy. Sequencing was performed on HiSeq2500. Mutations were detected in 7 (87.5%) of 8 participants. Six children had 8 mutations in the genes associated with congenital core myopathies; one patient had 2 mutations in the LAMA2 gene implicated in merosin-deficient muscular dystrophy. The proportions of patients with mutations in RYR1 and SEPN1 were equal (42.86%). Of 10 detected mutations, 3 had not been previously described, including c.7561G>A in RYR1, c.485C>A in SEPN1 and p.Cys1136Arg in LAMA2. The clinical and morphological features of core myopathies suggest that genetic causes of this group of disorders should not be limited to RYR1 and SEPN1 genes only. This necessitates the search for and the study of other genes implicated in congenital myopathies or myodystrophy using state-of-the-art molecular genetic tools.

Background Epilepsy with intellectual disability limited to females (Epileptic encephalopathy, early infantile, 9; EIEE9) is a rare early infantile epileptic encephalopathy characterized by an unusual X-linked inheritance: females with heterozygous mutations are affected, while hemizygous males are not. Case presentation We describe the clinical and molecular characteristics of 2 Russian patients with EIEE9 (females, ages 3 years and 7 years). In these patients seizures developed at the age of 3 years. Additionally, for our patients and for cases described in the literature we searched for a possible relationship between the type and localization of the mutation and the EIEE9 clinical phenotype. Conclusions We identified two novel PCDH19 mutations in EIEE9 patients: a missense mutation in exon 1 (c.1236C > A, p.Asp412Glu) and a frameshift in exon 3 (c.2386_2387insGTCT, p.Thr796fs). We conclude that the age of seizure onset and the presence of intellectual disability may depend not on the type and localization of PCDH19 mutations, but on the X-inactivation status. The study also highlights the need to screen for EIEE9 among young female epilepsy patients.

Non-invasive prenatal testing (NIPT) for aneuploidy on Chromosomes 21 (T21), 18 (T18) and 13 (T13) is actively used in clinical practice around the world. One of the limitations of the wider implementation of this test is the high cost of the analysis itself, as high-throughput sequencing is still relatively expensive. At the same time, there is an increasing trend in the length of reads yielded by sequencers. Since extracellular DNA is short, in the order of 140&ndash, 160 bp, it is not possible to effectively use long reads. The authors used high-performance sequencing of cell-free DNA (cfDNA) libraries that went through additional stages of enzymatic fragmentation and random ligation of the resulting products to create long chimeric reads. The authors used a controlled set of samples to analyze a set of cfDNA samples from pregnant women with a high risk of fetus aneuploidy according to the results of the first trimester screening and confirmed by invasive karyotyping of the fetus using laboratory and analytical approaches developed by the authors. They evaluated the sensitivity, specificity, PPV (positive predictive value), and NPV (negative predictive value) of the results. The authors developed a technique for constructing long chimeric reads from short cfDNA fragments and validated the test using a control set of extracellular DNA samples obtained from pregnant women. The obtained sensitivity and specificity parameters of the NIPT developed by the authors corresponded to the approaches proposed earlier (99.93% and 99.14% for T21, 100% and 98.34% for T18, 100% and 99.17% for T13, respectively).

Background Neuronal ceroid lipofuscinoses (NCLs) are a group of neurodegenerative disorders characterized by an accumulation of lipofuscin in the body's tissues. NCLs are associated with variable age of onset and progressive symptoms including seizures, psychomotor decline, and loss of vision. Methods We describe the clinical and molecular characteristics of four Russian patients with NCL (one female and three males, with ages ranging from 4 to 5 years). The clinical features of these patients include cognitive and motor deterioration, seizures, stereotypies, and magnetic resonance imaging signs of brain atrophy. Exome sequencing was performed to identify the genetic variants of patients with NCL. Additionally, we tested 6,396 healthy Russians for NCL alleles. Results We identified five distinct mutations in four NCL‐associated genes of which two mutations are novel. These include a novel homozygous frameshift mutation in the CLN6 gene, a compound heterozygous missense mutation in the KCTD7 gene, and previously known mutations in KCTD7, TPP1, and MFSD8 genes. Furthermore, we estimated the Russian population carrier frequency of pathogenic and likely pathogenic variants in 13 genes associated with different types of NCL. Conclusion Our study expands the spectrum of mutations in lipofuscinosis. This is the first study to describe the molecular basis of NCLs in Russia and has profound and numerous clinical implications for diagnosis, genetic counseling, genotype–phenotype correlations, and prognosis., 5 distinct mutations were identified in 4 NCL‐caused genes, of which two mutations are novel. Also carrier frequency of pathogenic and likely pathogenic variants in 13 genes associated with different types of NCL were estimated in Russian population.

Recent advances in DNA sequencing open prospects to make whole-genome analysis rapid and reliable, which is promising for various applications including personalized medicine. However, existing techniques for {\it de novo} genome assembly, which is used for the analysis of genomic rearrangements, chromosome phasing, and reconstructing genomes without a reference, require solving tasks of high computational complexity. Here we demonstrate a method for solving genome assembly tasks with the use of quantum and quantum-inspired optimization techniques. Within this method, we present experimental results on genome assembly using quantum annealers both for simulated data and the $\phi$X 174 bacteriophage. Our results pave a way for an increase in the efficiency of solving bioinformatics problems with the use of quantum computing and, in particular, quantum annealing. We expect that the new generation of quantum annealing devices would outperform existing techniques for {\it de novo} genome assembly. To the best of our knowledge, this is the first experimental study of de novo genome assembly problems both for real and synthetic data on quantum annealing devices and quantum-inspired techniques., Comment: 9 pages, 4 figures

Background Liddle syndrome is a monogenic disease with autosomal dominant inheritance. Basic characteristics of this disease are hypertension, reduced concentration of aldosterone and renin activity, as well as increased excretion of potassium leading to low level of potassium in serum and metabolic alkalosis. The cause of Liddle syndrome is missense or frameshift mutations in SCNN1A, SCNN1B, or SCNN1G genes that encode epithelial sodium channel subunits. Case presentation We describe a family with Liddle syndrome from Russia. 15-year-old proband has arterial hypertension, hypokalemia, hyporeninemia, metabolic alkalosis, but aldosterone level is within the normal range. At 12 years of age, arterial hypertension was noticed for the first time. We identified novel frameshift mutation c.1769delG (p.Gly590Alafs) in SCNN1G, which encodes the γ subunit of ENaC in vertebrates. The father and younger sister also harbor this heterozygous deletion. Treatment with amiloride of proband and his sister did not normalize the blood pressure, but normalized level of plasma renin activity. Conclusions Our results expand the mutational spectrum of Liddle syndrome and provide further proof that the conserved PY motif is crucial to control of ENaC activity. Genetic analysis has implications for the management of hypertension, specific treatment with amiloride and counselling in families with Liddle syndrome.

Background Neuronal ceroid lipofuscinoses (NCLs) are the most common autosomal recessive neurodegenerative disorders in children. Clinical manifestations include progressive cognitive decline, motor impairment, ataxia, visual loss, seizures and early death. To date more than 440 NCL-causing mutations in 13 genes are known. Case presentation We report clinical and genetic characteristics of a 5-year-old girl affected by ceroid lipofuscinosis type 7 (NCL7). She had progressive motor and mental deterioration since the age of 2,5 years. Later she developed progressive vision loss, stereotypies, action myoclonus and epilepsy. By the age of 5 years she stopped walking. Based on symptoms, diagnosis of Rett syndrome was suggested, but no abnormalities were detected in MeCP2. We identified a novel homozygous mutation in MFSD8 gene (c.525 T > A, p.Cys175Ter). To our knowledge, this is the first report of MFSD8 gene mutation in a Russian patient with variant late-infantile NCL. Conclusions Our results enlarge mutational spectrum of ceroid lipofuscinosis type 7 and demonstrate tremendous diagnosis value of exome sequencing for pediatric NCLs. Also we confirmed that NCL should be suspected in patients with Rett-like phenotype at onset and negative MECP2 mutation.

We aimed to develop a pipeline for the bioinformatic analysis and interpretation of NGS data and detection of a wide range of single-nucleotide somatic mutations within tumor DNA. Initially, the NGS reads were submitted to a quality control check by the Cutadapt program. Low-quality 3¢-nucleotides were removed. After that the reads were mapped to the reference genome hg19 (GRCh37.p13) by BWA. The SAMtools program was used for exclusion of duplicates. MuTect was used for SNV calling. The functional effect of SNVs was evaluated using the algorithm, including annotation and evaluation of SNV pathogenicity by SnpEff and analysis of such databases as COSMIC, dbNSFP, Clinvar, and OMIM. The effect of SNV on the protein function was estimated by SIFT and PolyPhen2. Mutation frequencies were obtained from 1000 Genomes and ExAC projects, as well as from our own databases with frequency data. In order to evaluate the pipeline we used 18 breast cancer tumor biopsies. The MYbaits Onconome KL v1.5 Panel ("MYcroarray") was used for targeted enrichment. NGS was performed on the Illumina HiSeq 2500 platform. As a result, we identified alterations in BRCA1, BRCA2, ATM, CDH1, CHEK2, TP53 genes that affected the sequence of encoded proteins. Our pipeline can be used for effective search and annotation of tumor SNVs. In this study, for the first time, we have tested this pipeline for NGS data analysis of samples from patients of the Russian population. However, further confirmation of efficiency and accuracy of the pipeline is required on NGS data from larger datasets as well as data from several types of solid tumors.Tsel'iu issledovaniia byla razrabotka i testirovanie protokola dlia bioinformaticheskoĭ obrabotki NGS-dannykh dlia éffektivnogo poiska mutatsiĭ v genome solidnykh opukholeĭ. Soglasno razrabotannomu protokolu na nachal'nom étape provodili otsenku kachestva prochteniĭ nukleotidov. Nukleotidy s kachestvom prochteniia nizhe 10 udaliali s 3¢-kontsa s pomoshch'iu programmy Cutadapt. Dalee prochteniia kartirovali na referensnyĭ genom hg19 (GRCh37.p13) s pomoshch'iu programmy BWA. Deduplikatsiiu ridov vypolniali spetsializirovannoĭ programmoĭ SAMtools. Dlia raspoznavaniia odnonukleotidnykh variantov primeniali MuTect. Kompleksno otsenivali funktsional'nyĭ éffekt mutatsiĭ na osnove algoritma, vkliuchaiushchego annotirovanie i otsenku patogennosti s pomoshch'iu programmnogo resheniia SnpEff, a takzhe analiza takikh baz dannykh, kak COSMIC, dbNSFP, Clinvar, OMIM. Dopolnitel'no dlia otsenki éffekta na funktsiiu kodiruemogo belka primeniali utility SIFT i Poly-Phen2. Informatsiiu o chastote mutatsiĭ poluchali na osnove dannykh proektov 1000 Genomes i ExAC, a takzhe sobstvennoĭ bazy dannykh chastot. Dlia provedeniia testirovaniia protokola byl proveden analiz 18 obraztsov biopsii opukholeĭ molochnoĭ zhelezy. Sekvenirovanie obraztsov provodili na platforme Illumina. Dlia targetnogo obogashcheniia kodiruiushchikh regionov genoma ispol'zovali nabor reagentov MYbaits Onconome KL v1.5 Panel (“MYcroarray,” SShA). Po rezul'tatam bioinformaticheskoĭ obrabotki dannykh sekvenirovaniia obnaruzheno mnozhestvo mutatsiĭ v genakh BRCA1, BRCA2, ATM, CDH1, CHEK2, TP53, v tom chisle mutatsii-draĭvery, okazyvaiushchie vliianie na aminokislotnuiu posledovatel'nost' kodiruemogo belka. Takim obrazom, predlagaemyĭ nami bioinformaticheskiĭ protokol pozvoliaet éffektivno vypolniat' avtomaticheskuiu obrabotku NGS-dannykh obraztsov opukholeĭ i obnaruzhivat' mutatsii. Dannyĭ protokol dlia bioinformaticheskoĭ obrabotki dannykh vpervye aprobirovan na vyborke obraztsov opukholeĭ iz rossiĭskoĭ populiatsii. Dlia podtverzhdeniia éffektivnosti i tochnosti predlagaemogo protokola v dal'neĭshem neobkhodimo testirovanie algoritma na dannykh sekvenirovaniia razlichnykh form opukholeĭ.

Depression is one of the leading causes of decreased quality of life and social functioning of patients. In the context of preventive medicine, the prevention of depression becomes a priority. To achieve the goals of prevention, it is necessary to identify specific population risk groups - individuals with a high genetic risk of depression. The paper describes the project aimed at developing a genetic test system based on polygenic risk scores (PRS) for depression, considering the multi-ethnicity and multicultural diversity of the Russian population. As a result of the study, data on the genetic architecture of depression (GWAS) and PRS for depression will be obtained for the first time. The emergence of a genetic test system developed in the study of the Russian population and in the conditions of a constant decrease in the cost of genetic research will allow an effective transition to preventive medicine in the area of mental health.Депрессия является одной из ведущих причин снижения качества жизни и социального функционирования пациентов. В рамках превентивной медицины профилактика депрессии становится приоритетным направлением. Для достижения задач профилактики необходимы возможности выявления в популяции групп риска — индивидуумов с высоким уровнем генетического риска развития депрессии. В статье описываются методология и дизайн проекта, направленного на разработку генетической тест-системы на основе полигенных шкал риска (PRS) развития депрессии с учетом полиэтничности и мультикультурального характера российской популяции. В результате выполнения исследования должны быть впервые получены данные о генетической архитектуре депрессии (методом GWAS) и выявлены полигенные шкалы риска (PRS) развития депрессии. Появление генетической тест-системы, разработанной при изучении российской популяции и в условиях постоянного снижения стоимости генетических исследований, позволит осуществить эффективный переход к развитию превентивной медицины в области психического здоровья.