Chordoma associated with tuberous sclerosis complex (TSC) is an extremely rare tumor that was described only in 13 cases since 1975. Сhordoma itself is a malignant slow-growing bone tumor thought to arise from vestigial or ectopic notochordal tissue. Chordoma associated with TSC differs from chordoma in the general pediatric population in the median age, where the diagnosis of TSC-associated chordoma is 6.2 months, whereas for chordoma in the general pediatric population it is set to 12 years. The majority of TSC-associated chordomas are localized in skull-based and sacrum regions, and rare in the spine. Chordomas are genetically heterogeneous tumors characterized by chromosomal instability (CIN), and alterations involving PI3K-AKT signaling pathway genes and chromatin remodeling genes. Here we present the 14th case of chordoma associated with TSC in a 1-year-old pediatric patient. Alongside biallelic inactivation of the TSC1 gene, molecular genetic analysis revealed CIN and involvement of epigenetic regulation genes. In addition, we found the engagement of CBX7 and apolipoprotein B editing complex (APOBEC3) genes that were not yet seen in chordomas before. Amplification of CBX7 may epigenetically silence the CDKN2A gene, whereas amplification of APOBEC3 genes can explain the frequent occurrence of CIN in chordomas. We also found that KRAS gene is located in the region with gain status, which may suggest the ineffectiveness of potential EGFR monotherapy. Thus, molecular genetic analysis carried out in this study broadens the horizons of possible approaches for targeted therapies with potential applications for personalized medicine.

The level of tumor mutation burden (TMB) is a predictive factor of immune checkpoints that determines the potential effectiveness of immune checkpoint inhibitors and indications for their prescription regardless of the type of tumor in adults and children. However, the prevalence of tumors with high TMB in the pediatric population has not been well studied. Objective of the research: to assess the detection frequency of high TMB (>10 mutations per megabase) in pediatric hepatocellular carcinoma (HCC). Materials and methods of research: Next Generation Sequencing, Ion AmpliSeq™ Comprehensive Cancer Panel (409 genes) of tumor DNA samples from 4 children with HCC was performed. The calculation of the mutational load was carried out according to the work of Z.R. Chalmers et al. Results: out of 4 analyzed samples, TMB levels have lower cut-off value than needed for the immunity checkpoint inhibitors for 2 patients to administer: 5,9 mut/MB (primary tumor), 9,2 mut/MB (metastasis), while in 2 other patients the TMB has level above the threshold – 10,9 mut/MB (primary tumor) and 48,5 mut/MB (relapse metastasis), respectively. Conclusion: this paper presents the initial experience of estimation of the TMB level in children with HCC. In our case series report, the level allowing the prescription of immunotherapy (>10 mut/MB) was observed in 2 patients. Further research on a larger cohort of patients is useful to assess the role of mutational burden in disease prediction and effectiveness of immunotherapy.

Синдром Сотоса представляет собой редкое аутосомно-доминантное заболевание с частотой встречаемости около 1:14000 новорожденных. Несмотря на ряд характерных клинических проявлений, его диагностика затруднена в связи с высокой степенью перекрывания фенотипа с рядом других наследственных синдромов, таких как синдром Видемана-Беквита или синдром Марфана. Молекулярно-генетической причиной синдрома Сотоса являются мутации в гене NSD1, приводящие к дисфункции белка. В статье представлены и охарактеризованы варианты в гене NSD1, найденные в ходе диагностики методами высокопроизводительного параллельного секвенирования (NGS) и MLPA. Sotos syndrome is a rare autosomal dominant disorder with an incidence of about 1 in 14,000 newborns. Despite a number of characteristic clinical manifestations, its diagnosis is difficult due to the high degree of overlap of the phenotype with a number of other hereditary syndromes, such as Beckwith-Wiedemann syndrome or Marfan syndrome. The molecular genetic cause of Sotos syndrome is mutations in the NSD1 gene, leading to protein dysfunction. The article presents and characterizes variants in the NSD1 gene found during diagnostics using high-throughput sequencing (NGS) and MLPA.

Aim . To search for new pharmacogenetic biomarkers of bleeding risk in patients taking rivaroxaban and dabigatran for different indications: atrial fibrillation, endoprosthesis of large joints of lower limbs. Material and methods . The study enrolled 29 patients (17 patients received dabigatran and 12 –rivaroxaban), who had hemorrhagic complications during taking direct oral anticoagulants. To find new pharmacogenetic biomarkers of bleeding risk, a next generation sequencing (NGS) was performed for selected candidate genes. Results . Among the patients with bleeding who received dabigatran, 13 variants of the nucleotide sequence showed statistically significant deviation from the population values: 11 in the CES1 gene and 2 in the ABCB1 gene. Among the patients with bleeding who received rivaroxaban, 7 variants of nucleotide sequence showed significant deviation: 4 in the ABCG2 gene, 2 in the CYP3A4 gene, and 1 in the ABCB1 gene. Conclusion . The identified in this study polymorphisms of candidate genes ABCB1, ABCG2, CES1, CYP3A4 were associated with the risk of bleeding in patients taking rivaroxaban and dabigatran. It makes an important contribution to the pharmacogenetics of direct oral anticoagulants and require additional assessment of clinical significance in further studies.

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.

Despite the advantages of neoadjuvant chemotherapy (NACT), associated toxicity is a serious complication that renders monitoring of the patients’ response to NACT highly important. Thus, prediction of tumor response to treatment is imperative to avoid exposure of potential non-responders to deleterious complications. We have performed genome-wide analysis of DNA methylation by XmaI-RRBS and selected CpG dinucleotides differential methylation of which discriminates luminal B breast cancer samples with different sensitivity to NACT. With this data, we have developed multiplex methylation sensitive restriction enzyme PCR (MSRE-PCR) protocol for determining the methylation status of 10 genes ( SLC9A3, C1QL2, DPYS, IRF4, ADCY8, KCNQ2, TERT, SYNDIG1, SKOR2 and GRIK1 ) that distinguish BC samples with different NACT response. Analysis of these 10 markers by MSRE-PCR in biopsy samples allowed us to reveal three top informative combinations of markers, (1) IRF4 and C1QL2 ; (2) IRF4, C1QL2 , and ADCY8 ; (3) IRF4, C1QL2 , and DPYS , with the areas under ROC curves (AUCs) of 0.75, 0.78 and 0.74, respectively. A classifier based on IRF4 and C1QL2 better meets the diagnostic panel simplicity requirements, as it consists of only two markers. Diagnostic accuracy of the panel of these two markers is 0.75, with the sensitivity of 75% and specificity of 75%.

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.

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.

The human chromosome region 14q32 contains a number of imprinted genes that are expressed either from the paternal or from the maternal alleles. Genetic alterations of these genes lead to distinct phenotypes, known as maternal uniparental disomy 14 (Temple syndrome ОMIM #616222) or paternal uniparental disomy 14 (Kagami-Ogata syndrome ОMIM# 608149). Kagami-Ogata syndrome characterizes by a small bell-shaped thorax with a coat-hanger configuration of the ribs, abdominal wall defects, joint contractures and polyhydramnios during the pregnancy. In both syndromes, three types of molecular alterations have been reported: uniparental disomy 14, deletions and epimutations. Most described deletions in previous articles included one or both of the differentially methylated regions: DLK1 and MEG3. In contrast to uniparental disomy and epimutations, deletions affecting regulatory elements in 14q32.2 are associated with a high-recurrence risk. Patients and Methods. We present clinical case Kagami-Ogata syndrome is caused deletion 14q32.2. We performed microsatellite analysis of trio and microarray analysis of the mother. Results. Microsatellite analysis of trio and microarray analysis of mother have allowed to define the extent of deletions (378 kb) affecting the regions 14q32.2-14q32.1, including genes (MEG3, RTL1, MEG8) that are expressed from the maternal allele and do not affect the IG-DMR. We performed a comparative analysis of patient’s phenotypes, described earlier, with different deletions involving IG-DMR and MEG3-DMR. Conclusion. Analysis of deletions origin, as well as its included genes, in the diagnosis of diseases allows making an accurate diagnosis and using the methods of prenatal and/or preimplantation diagnostics in patients with a high risk of hereditary pathology.

Background: Usher syndrome (USH) is heterogeneous in nature and requires genetic test for diagnosis and management. Mutations in USH associated genes are reported in some populations except Russians. Here, we first time represented the mutation spectrum of a Russian USH cohort. Methods: Twenty-eight patients with USH were selected from 3214 patients from Deaf-Blind Support Foundation "Con-nection" during 2014-2016 following the observational study NCT03319524. Complete ophthalmologic, ENT, and vestibular medical tests were done for clinical characterization. NGS, MLPA, and Sanger sequencing were considered for genetic analysis. Results: Around 53.57% and 39.28% patients had USH1 and USH2, respectively; 17.85% cases (n = 5/28) had no known mutation. Eleven (73.33%) subjects showed variations in USH1 associated genes MYO7A (72.72%), CDH23 (9.09%), PCDH15 (9.09%), and USH1C (9.09%). Eleven mutations are detected in MYO7A where 54.54% are novel. MYO7A: p.Q18* was most frequent (27.27%) mutation and is associated with early manifestation and most severe clinical picture. Two novel mutations (p.E1301* and c.158-?_318+?del) are detected in PCDH15 gene. Around 90.90% patients suspected to be USH2 are confirmed by genetic testing. Eleven mutations detected in the USH2A gene, where 27.27% were novel. Most common USH2A mutation is p.W3955* (50%) followed by p.E767fs, p.R1653*, and c.8682-9A> G (20% each). Conclusion: The Russian USH cohort shows both novel and known USH mutations. Clinically the prevalence of USH2 is low (39.28%) and the frequency of MYO7A mutations responsible for USH1B is very high (63.63%, N = 7/11) compared to other cohorts. These seven patients carrying MYO7A mutations are preliminarily eligible for the UshStat (R) gene therapy.

Background. The use of high-throughput parallel sequencing (NGS) is fraught with errors: not all of the genetic variants that are detected by NGS are true and are confirmed by alternative methods. Incorrectly mapped reads contribute to the appearance of false-positive variants. We believe that for targeted sequencing using Ion AmpliSeq technology, the task of excluding erroneously mapped reads can be solved algorithmically. Additional information on the genomic coordinates of target regions and primers used in amplification allows us to evaluate the validity of the mapping of each reading and to exclude readings from the further analysis that do not correspond to the design of the experiment. Objective. To develop an algorithm to minimize the contribution of mapping errors to the spectrum of genetic variants detected by NGS. Material and methods. Using the information on the genomic coordinates of target regions and primers, we have analyzed 30 BAM files obtained by Ion AmpliSeq (targeted multiplex PCR) NGS with commercial Ion AmpliSeq Comprehensive Cancer Panel (15,992 target regions) and Ion AmpliSeq Inherited Disease Panel (10,309 target regions). The algorithm for excluding incorrectly mapped reads was implemented using the Python programming language. Result. We have performed comparison of the initial set of reads and the set obtained after excluding incorrectly mapped reads. This comparison revealed three groups of genetic variants: (1) detectable in both sets of reads, 6072 variants; (2) detectable exclusively in the original set, 127 (the predominant part of these variants is present in most samples and can be interpreted as a result of systematic alignment errors); (3) detectable in the set generated by exclusion of erroneously mapped reads only, 63 (true positive, previously masked variants). Conclusion. The use of additional information on the expected start and end of the read in the targeted study allows to (1) reduce the number of false-positive genetic variants detected due to misleading reads, (2) detect new ones that were not previously detected due to a seemingly low allele frequency, (3) obtain more reliable values of allelic frequencies of the identified variants. The use of our algorithm to exclude the incorrect mapping of the of DNA fragment reads increases the quality of interpretation of the NGS results, which is especially important for DNA diagnostics.

OBJECTIVE:FBLN5 encodes a key protein of elastic fiber matrix assembly and function that contributes to maintaining pelvic support and plays the important role in the pathophysiology of pelvic organ prolapse (POP). The aim of the study was to investigate whether there is an association between common single-nucleotide polymorphisms (SNPs) of the FBLN5 gene and POP. STUDY DESIGN:A total of eleven tag SNPs of the FBLN5 gene were genotyped using the polymerase chain reaction with confronting two-pair primers (PCR-CTPP) in 210 patients with POP (stages III-IV) and 292 controls with no even minimal POP. RESULTS:We revealed significant associations of tag SNPs rs2018736 and rs12589592 with POP. The top association signal was found for SNP rs2018736 (protective effect for the minor allele A) in the entire set: p=0.0026, OR=0.42, 95% CI: 0.24-0.75; in the stratum with pelvic floor trauma: p=0.0018, OR=0.27, 95% CI: 0.11-0.64; and in the stratum with fetal macrosomia: p=0.013, OR=0.14, 95% CI: 0.03-0.71. The results of the haplotype analyses were consistent with the single SNP analysis. In the strata without perineal trauma and fetal macrosomia effects were non-significant, possibly, due to the smaller effect sizes. CONCLUSIONS:Current data provide, for the first time, strong evidence that common SNPs of the FBLN5 gene are associated with POP especially after pelvic floor injury.

Abstract Oxidative stress may play a role in the recurrent miscarriage (RM) with no known etiology. This study was conducted to investigate the association of polymorphisms in oxidative stress-related genes with idiopathic RM. A total of 331 idiopathic RM patients and 197 controls were genotyped for ABCB1 rs1045642, CYP1A1 rs1048943 and rs4646903, COMT rs4680, CAT rs17880664, GCLC rs17883901, GPX4 rs713041, NRF2 rs6721961, SOD2 rs4880, and OGG1 rs1052133. A protective effect of COMT rs4680-G allele on RM was shown in individual SNP analysis: P = 0.0016, OR = 0.47, 95% CI 0.29–0.75. The multi-factor dimensionality reduction (MDR) approach revealed gene–gene interactions for ABCB1, COMT, GPX4, and OGG1 genes. Cumulative gene risk score analysis demonstrated that more than three risk alleles in the genes ABCB1 (rs1045642-T), COMT (rs4680-A), GPX4 (rs713041-T), and OGG1 (rs1052133-G) were associated with idiopathic RM P = 1.2 × 10− 3, OR = 1.97, 95% CI 1.31–2.97. In silico data interpreting by GeneMANIA analysis revealed genetic, physical, pathway, and coexpression networks for these four genes.