[Determination of the Subclonal Tumor Structure in Childhood Acute Myeloid Leukemia and Acral Melanoma by Next-Generation Sequencing].

Intratumoral heterogeneity and clonal variability are among the central problems of clinical oncology, leading to resistance to therapy, relapse, and metastasis. High-throughput sequencing of the tumor exome makes it possible to investigate the subclonal tumor organization. Target panel, clinical exome, and complete exome sequencing data were compared in tumors with different mutational burden, acute myeloid leukemia (AML) in children and acral melanoma. Targeted sequencing of AML samples detected more than one potential driver mutation in the signaling pathway genes KIT, NRAS, KRAS, CBL, and FLT3 in one patient, reflecting the complex clonal structure of the tumor substrate. Clusters of mutant alleles corresponding to different populations of leukemic cells in a sample were isolated based on exome sequencing data from the same AML patients. A comparison of the mutation profile for a primary AML sample and samples obtained in remission and relapse made it possible to trace the dynamic changes in the clonal composition of the tumor. The subclonal tumor structure was investigated in an acral melanoma case as an example. Mutant alleles present in the sample with close frequencies were clustered using the SciClone and ClonEvol packages. The results were used to predict the intratumoral clonal composition and to assume a clonal evolution model, which described the changes in the clonal composition of the tumor during metastasis, including the appearance of new mutations that might be associated with further disease progression. The approach used in the work is suitable for identifying the mutations that cause the formation of new tumor clones, which may have a proliferative advantage, in particular, in conditions of antitumor therapy.

[Monitoring of nocturnal penile tumescence in healthy volunteers by the Androscan MIT registrar to establish reliable normal physiological values in a multicenter study].

AIM: To evaluate the normal numerical and graphic values of nocturnal penile tumescence (NPT) test using Androscan MIT in healthy males in order to use the collected data as reference standard. MATERIALS AND METHODS: NPT monitoring was carried out in 38 healthy male volunteers by fixing a sensor designed for 20 measurements on their penis. During the NPT test the following parameters were recorded: 1) total sleep time; 2) minimal penis diameter (PD) in the flaccid state recorded by the apparatus; 3) maximum PD during effective penile tumescence; 4) absolute PD increase during effective erections; 5) increase in PD in %; 6) the overall time of the rigid-erection phase; 7) the number of penile rigidity episodes; 8) the average duration of each effective erection; 9) the percentage of rigid erections during the whole monitoring period. RESULTS: Based on the data collected by Androscan MIT, we have specified the numerical values which characterize normal physiological NPT indices in healthy male volunteers. The number of tests performed and similar inclusion criteria contribute to the objectiveness of the presented data. Normal sleep time was from 7.3 to 9.5 hours. Monitoring during sleeping reflected the upper and lower limits of the minimal PD value recorded by Androscan MIT, which was from 23.5 mm to 30.2 mm. The maximum PD increase during the most effective erection varied from 35.3 mm to 44.3 mm. Total PD increase was from 10.6 mm to 15.4 mm (from 35.6% to 59.2%). In all cases a significant difference in PD increase between flaccid state and effective erection during sleeping was seen. The number of penile rigidity episodes varied from 3 to 7 a night. The overall time of effective erections was from 62.3 to 206.7 minutes, while the minimum duration of a single erection episode was 16.4 minutes and the maximum duration reached 35.8 minutes. The ratio of effective NPT to the total sleep time (the percentage of penile rigidity episodes) varied from 11.9% to 41.3%. CONCLUSION: Our results allowed to define reference qualitative and quantitative values of NPT in healthy male volunteers recorded by Androscan MIT which can be considered as normal and physiological and used for differential diagnostics of erectile dysfunction.

[Germline and Somatic Mutations in Archived Breast Cancer Specimens of Different Subtypes].

Molecular profiling of tumors may provide promising options for personalized treatment. We have examined the spectrum of germline and somatic mutations in 23 breast cancers (ВС) of various molecular subtypes, including tumors 1) with expression of estrogen, progesterone and/or epidermal growth factor receptor HER2/neu, and 2) with a triple negative phenotype. Genomic DNA specimens were isolated from archived tumor and normal tissue samples and subjected to targeted sequencing of the coding regions of 25 cancer-associated genes with a mean coverage of x 1000. In the triple negative subtype of ВС, the pathogenic germline mutations BRCA1 c.66_67delAG (185delAG) and BRCA1 c.3226_3227AG (3347delAG) were detected, while the germline mutation BRCA2 658_659del (886delGT) was found in patients with positive receptor staining. Mutations in BRCAl/2 were overrepresented by frequency (80%), pointing at common loss of heterozygosity affecting the normal allele. Somatic mutations in the TP53 gene were found in 7/10 (70%) patients with the triple negative subtype of ВС and in 3/13 (23%) in the group with positive receptor staining. Additionally, in both groups of patients, somatic mutations of the PTEN, MSH2, MSH6, and MUTYH genes were detected.

[Driver Mutations in Acute Myeloid Leukemia with Inversion of Chromosome 16].

Certain subtypes of acute myeloid leukemia occur as a result of the cooperation of several events these are, the formation of fusion genes as a result of chromosomal rearrangements, which leads to the disruption of cell differentiation, and the emergence of mutations that enhance cellular proliferation by activating intracellular signaling pathways. High-throughput sequencing methods reveal characteristic mutation spectra in leukemia associated with different chromosomal disorders. However, the role of mutation events in malignant cell transformation processes remains obscure. We searched for driver mutation events in leukemic cells containing the chimeric CBFB-MYH11 gene, which results from inversion of chromosome 16. Using target enrichment, the coding regions of 84 genes in genomes of 12 children with acute myeloid leukemia with inv(16) were investigated. Somatic mutations have been found in the genes of the proteins of intracellular signaling cascades mediated by receptor tyrosine kinases, such as KIT (41%), NRAS (25%), KRAS (17%), and FLT3 (8.3%). Comparative analysis of samples at the time of diagnosis and during remission was used to assess the role of mutations in the pathogenesis of the disease. Previously undescribed mutations in the KDM6A, NOTCH1, and IDH1 genes, which may be involved in leukemogenesis processes have been identified.

[Somatic Mutations Associated with Metastasis in Acral Melanoma].

Acral melanoma is one of the most aggressive and fast-growing forms of cutaneous melanoma and is characterized by a predominant location on the palms and feet. Primary tumors, metastases, and normal tissue samples from five acral melanoma patients were examined by massive parallel sequencing, focusing on the coding regions of 4100 genes involved in the origin and progression of hereditary and oncology diseases. Somatic mutations were found in genes related to cell division, proliferation, and apoptosis (BRAF, NRAS, VAV1, GATA1, and GCM2); cell adhesion (CTNND2 and ITGB4); angiogenesis (VEGFA); and the regulation of energy metabolism (BCS1L). Comparisons of target DNA sequences between morphologically normal and primary tumor tissues and between normal and metastatic tissues identified the candidate genes responsible for rapid metastasis in acral melanoma.

[Mutational Profiling of Pediatric Myeloid Leukemia Subtypes without Clinically Significant Chromosomal Aberrations].

The discovery of novel significant molecular and genetic markers is important for the diagnostics, prognosis, and therapy selection in hematological malignancies. Distinct cytogenetic aberrations leading to the formation of fusion genes are found in more than 40% of pediactric cases of acute myeloid leukemia (AML); however, the tumor cells in approximately 20% of these patients display cytogenetically normal karyotype (NK-AML). Here we present the analysis of the mutational profiles of leukemic cells collected from pediatric AML cases without known clinically significant chromosomal aberrations aimed at identifying AML specific markers. In 34 pediatric cases of different AML types, the coding regions of 26 genes involved in the AML pathogenesis were analyzed by massive parallel sequencing. Sequencing revealed the somatic mutations in genes that are involved in various intracellular signaling pathways, including the CEBPA, ETV, IDH1, JAK2, and NRAS genes. In addition, rare genetic variants were found in CUX1, FLT3, TET2, PTPN11, and NUP98 genes. This data may contribute to the understanding of the mechanisms of malignant cell transformation in the case of leukemogenesis.

[A clinical and genetic analysis of risk factors for the development of acute and chronic cerebral ischemia]. / Kliniko-geneticheskii analiz faktorov riska razvitiia ostroi i khronicheskoi ishemii golovnogo mozga.

To study the association between polymorphic markers in the ACE, SERPINE1, FGB, F5, F7, F12, GP1BA, GPIIIa, MTHFR, CYP11B2, PON1, PON2, NOS2, NOS2, HIFla, LTA, ALOX5AP genes and clinical characteristics of acute and chronic forms of circulatory disorders of the brain. MATERIAL AND METHODS: The analysis of polymorphic variants in ACE, FGB, F5, F7, F12, GP1BA, GPIIIa, SERPINE1, MTHFR, CYP11B2, PON1, PON2, NOS2, NOS3, PDE4D, HIF1a, LTA, ALOX5AP in 81 patients with chronic cerebral ischemia (CCI) and 69 patients with ischemic stroke (IS), and their interrelation with clinical manifestations of disease were investigated. RESULTS AND CONCLUSION: The association between the T/T genotype of the PDE4D SNP 83C>T polymorphism and a rapid progression of hypertensive disease (GB) was revealed (OR=6.22, CI=1.86-20.79, p=0.0036) in the group of patients with CCI. The association of the allele D and the DD genotype of the ACE (I>D, rs1799752) with cardioembolic stroke (OR=2.67, 95% CI=1.23-5.8, p=0.02 and OR=7.14, 95% CI=1.72-29.69, p=0.0057) was found. When comparing subgroups of patients with different degrees of stenosis of brachiocephalic arteries (BCA), the association of the allele C and the TC genotype of the GP1BA (rs2243093, -5T/C) with BCA occlusion and expressed hemodynamically significant stenosis (>75%) was revealed (OR=3.39, 95% CI=1.12-10.25, p=0.03 and OR=4.44, 95% CI=1.27-15.54, p=0.023, respectively). Thus, polymorphic markers in PDE4D, ACE, GP1BA in combination with certain clinical characteristics are risk factors for the progression of CCI and development of IS.

[Relationships of rs7341475 polymorphism and DNA methylation in the reelin gene with schizophrenia symptoms]. / Sviaz' polimorfizma rs7341475 i metilirovaniia DNK v gene rilina s proiavleniiami shizofrenii.

AIM: To study the role of polymorphism rs7341475 and methylation of the reelin gene in symptoms of schizophrenia and semantic verbal fluency. MATERIAL AND METHODS: Genotypes at the locus rs7341475 were identified in 556 patients with schizophrenic disorders. PANSS scores were obtained for 549 patients and 221 patients performed a test for semantic verbal fluency. The association of the reelin promoter methylation with the PANSS and verbal fluency measures was evaluated in 35 patients. A five-factor model of the PANSS was used. RESULTS: The interaction effect of sex with genotype on the PANSS scores was found (F=2.70, p=0.020). Schizophrenic men homozygous for a common allele G had the lowest scores of the positive syndrome. Verbal fluency was related to the reelin promoter methylation. CONCLUSION: The results suggest that polymorphism rs7341475 may be associated with the variability of positive symptomatology in schizophrenic men. At the same time, the reelin gene methylation pattern, which consists of a higher methylation level in the region of the transcription start site and a lower one in the distal region of the promoter, may be beneficial for verbal fluency.

[Oncolytic Properties of a Mumps Virus Vaccine Strain in Human Melanoma Cell Lines].

The oncolytic potential of the attenuated mumps virus (MV) vaccine strain Leningrad-3 (L-3) was evaluated in a panel of four human metastatic melanoma cell lines. The lines were shown to be susceptible and permissive to MV infection. Efficient MV replication led to death of melanoma cells, but the effect differed among the cell lines. Possible mechanisms mediating the selectivity of MV L-3 towards the cell lines were explored. Replicative and oncolytic activity of MV was found to depend on the expression pattern of type I interferon genes. None of the melanoma cell lines showed induction of expression of the total spectrum of genes required to inhibit virus replication. Based on the results, MV L-3 was assumed to be a promising oncolytic agent for human melanoma cells.

[Substrate Properties of New Fluorescently Labeled Deoxycytidine Triphosphates in Enzymatic Synthesis of DNA with Polymerases of Families A and B].

The efficiency of the incorporation of fluorescently labeled derivatives of 2'-deoxycytidine in DNA synthesized de novo has been studied using PCR with Taq and Tth polymerases of family A and Vent (exo-) and Deep Vent (exo-) polymerases of family B. Four derivatives of 5'-triphosphate-2'-deoxycytidine (dCTP) have different chemical structures of the indodicarbocyanine dye and Cy5 analogue attached to position 5 of cytosine. The kinetics of the accumulation of the PCR products and the intensity of the fluorescent signals in the hybridization analysis with immobilized DNA probes depend on the modification of the fluorescently labeled dCTP counterpart, its concentration, and the type of DNA polymerase. All labeled triphosphates showed some inhibitory effects on PCR. The best balance between the efficiency of incorporating labeled cytidine derivatives and the negative effect on the PCR kinetics has been shown in the case of Hot Taq polymerase in combination with the Cy5-dCTP analogue, which contains containing electrically neutral chro-mophore, the axis of which is a continuation of the linker between the chromophore and the pyrimidine base.

[Identification of Fusion Transcripts in Leukеmic Cells by Whole-Transcriptome Sequencing].

Genetic aberrations in leukemia often lead to the formation of expressed chimeric genes, which should be assessed for proper diagnosis and therapy. Modern methods of molecular diagnostic mainly allow to identify already known fusion genes. RNAseq is an efficient tool for identification of rare and novel chimeric transcripts. Here we present the results of the whole transcriptome analysis of bone marrow samples from five patients with acute myeloblastic leukemia and one, with myelodysplastic syndrome. The whole-transcriptome analysis was performed using Illumina/Solexa approach. We found rare or unknown chimeric transcripts including ETV6-MDS1, MN1-ETV6, OAZ1-PTMA, and MLLT10-GRIA4. Each of these transcripts was confirmed by RT-PCR and Sanger sequencing.

[Multiplex Genotyping of Allelic Variants of Genes Involved in Metabolizing Antileukemic Drugs].

A biochip, primer set, and genotyping protocol were developed to simultaneously address 16 single nucleotide polymorphisms in antileukemic drug metabolism genes, including TPMT, ITPA, MTHFR, SLCO1B1, SLC19A1, NR3C1, GRIA1, ASNS, MTRR, and ABCB1. The genotyping procedure included a one-round multiplex polymerase chain reaction (PCR) with simultaneous incorporation of a fluorescent label into the PCR product and subsequent hybridization on a biochip with immobilized probes. The method was used to test 65 DNA samples of leukemia patients. Fluorescence signal intensity ratios in pairs of wild-type and respective mutant sequence probes were analyzed for all polymorphic markers and demonstrated high accuracy of genotyping. The reliability of genotype determination using the biochip was confirmed by direct Sanger sequencing.

The EIMB Hydrogel Microarray Technology: Thirty Years Later.

 Biological microarrays (biochips) are analytical tools that can be used to implement complex integrative genomic and proteomic approaches to the solution of problems of personalized medicine (e.g., patient examination in order to reveal the disease long before the manifestation of clinical symptoms, assess the severity of pathological or infectious processes, and choose a rational treatment). The efficiency of biochips is predicated on their ability to perform multiple parallel specific reactions and to allow one to study the interactions of biopolymer molecules, such as DNA, proteins, glycans, etc. One of the pioneers of microarray technology was the Engelhardt Institute of Molecular Biology of the Russian Academy of Sciences (EIMB), with its suggestion to immobilize molecular probes in the three-dimensional structure of a hydrophilic gel. Since the first experiments on sequencing by hybridization on oligonucleotide microarrays conducted some 30 years ago, the hydrogel microarrays designed at the EIMB have come a long and successful way from basic research to clinical laboratory diagnostics. This review discusses the key aspects of hydrogel microarray technology and a number of state-ofthe-art approaches for a multiplex analysis of DNA and the protein biomarkers of socially significant diseases, including the molecular genetic, immunological, and epidemiological aspects of pathogenesis.

[Multiplex Assay to Evaluate the Genetic Risk of Developing Human Melanoma].

A genotyping procedure based on single-step PCR and subsequent allele-specific hybridization on a hydrogel biochip was developed to address the polymorphisms of HERC2, OCA2, SLC24A4, SLC45A2, TYR, IRF4, MC1R,MITF, PIGU, MYH7B, NCOA6, and CDK10. Amplified gene fragments were fluorescently labeled in PCR, and fluorescent signals from biochip cells were detected to evaluate how efficiently the PCR product formed a perfect duplex with an immobilized probe. The analytical characteristics of hybridization analysis were estimated for several fluorophores with different optical spectra. Cyanine dyes fluorescing in the range of Cy5 and Cy7 were synthesized for the purpose and used as 5'-tags of universal primers in single-step PCR. A Cy7 analog fluorescing in the near infrared range was found to increase the sensitivity of hybridization analysis by producing a lower background signal in the cases where target gene amplification was low.

[Allelic variants of immune response genes in children with infectious complications during the treatment of acute leukemia].

Infectious complications that arise during the treatment of children with acute leukemia with chemotherapeutic agents at high doses represent a serious problem in oncohematology. To find genetic conditions that may lead to the development of postchemotherapy infections, the genomes of 12 children with acute leukemia who had severe infectious complications during therapy were examined. At the same time, the coding regions of 17 genes involved in the regulation of the immune response were determined by massive parallel sequencing. The analysis revealed 39 nonsynonymous SNPs that lead to amino acid substitutions, including the following informative genetic markers: PTPN22 c.1858C>T (rs2476601), TLR4 c.896A>G (rs4986790) and TLR4 c.1196C>T (rs4986791), IL7R c.197T>C (rs1494555) and IL7R c.412G>A (rs1494558). The results of massive parallel sequencing were validated by Sanger sequencing. The identification of genetic markers associated with the predisposition to infectious complications may allow one to assess the individual risk of the severe infection development in children with acute leukemia during the treatment with chemotherapeutic agents and to begin the development of personalized approaches to anticancer therapy.

[Detection of BCR-ABL gene mutations in chronic myeloid leukemia using biochips].

A biochip-based method was developed to identify the BCR-ABL mutations that affect the thyrosine kinase domain and determine resistance to targeted therapy with thyrosine kinase inhibitors. The method is based on RT-PCR followed by allele-specific hybridization on a biochip with immobilized oligonucleotide probes. The biochip addresses 11 mutations, which are responsible for up to 85% of imatinib resistance cases. A method to decect the clinically significant mutation T315I was designed on the basis of LNA-clamped PCR and proved highly sensitive, detecting the mutation in clinical samples with a leukemic cell content of 5% or higher. The method was validated using clinical samples from chronic myeloid leukemia (CML) patients with acquired resistance to imatinib. The results of hybridization on biochip were verified by Sanger sequencing.

[Biological microchip for establishing the structure of fusion transcripts involving MLL in children with acute leukemia].

MLL is involved in fusion genes with more than 100 partner genes, approximately 80 of which have been characterized at the molecular level. MLL fusion genes are often found in infants (60-80% of acute lymphoblastic leukemia (ALL) cases and 40-50% of acute myeloblastic leukemia (AML) cases) and are appreciably rarer (8-10%) in children older than 1 year of age. MLL rearrangements are important markers in diagnosis and treatment choice. To identify the partner gene is of primary importance for prognosis and minimal residual disease monitoring. The structure of the fusion gene, including localization of the MLL breakpoints, is also informative. A method was developed to examine the fusion transcripts in order to identify the partner gene among the six most common ones and to establish the exon structure of the rearranged MLL. The method includes a multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) to amplify and to fluorescently label a fusion transcript fragment and subsequent hybridization of the product on a biological microchip with immobilized oligonucleotides complementary to exons of MLL and its partner genes AFF1, MLLT1, MLLT3, MLLT4, MLLT10, and ELL. Hybridization results were verified by sequencing the RT-PCR products and, in some cases, performing long-distance inverse PCR (LDI-PCR). The study involved 38 bone marrow samples from ALL patients (including 33 children younger than 1 year of age) and 15 samples from AML patients (including 10 from children younger than 1 year of age). The main partner genes were AFF1 (49%), MLLT1 (27%), MLLT3 (12%), and MLLT10 (12%) in ALL and MLLT3 (80%), MLLT10 (10%), and MLLT4 (10%) in AML. Fusion gene transcripts most commonly included MLL exon 11 (58% of ALL cases and 50% of AML cases), suggesting a breakpoint in MLL intron 11.

[Brain neurotransmitter systems gene Polymorphism: the Search for pharmacogenetic markers of efficacy of haloperidol in Russians and Tatars].

Antipsychotics are the main drugs for the treatment of severe mental illness--schizophrenia affects about 1% of the population. The mechanism of action of neuroleptics is still up to the end. Several studies in the field of pharmacogenetics confirm enourmous influence of several neurotransmitter systems in the brain on the efficiency and the development of side effects. In this paper, we analyzed the association of nine polymorphic variants of five genes of dopaminergic and serotonergic systems DRD4, HTR2A, TPH1, SLC18A1, COMT in Russian and Tatars patients living in the Republic of Bashkortostan (RB) with the efficiency of a typical antipsychotic haloperidol on the scale of positive and negative systems of PANSS. The study established pharmacogenetic markers of increased and decreased effectiveness of therapy with haloperidol in the treatment groups. The results of this study confirm the importance of changes in the nucleotide sequences of the studied genes of the serotoninergic and dopaminergic systems (HTR2A, TPH1, SLC18A1 COMT, DRD4) in the formation of individual sensitivity to haloperidol. The results of our work considered as preliminary contact, requires an increase in the number of samples studied.

[Infrared fluorescent markers for microarray DNA analysis on biological microchip].

To expand the informational capabilities of molecular genetic research, on the biological microchips, new indotricarbocyanine dyes that fluoresce in the near infrared (IR) spectral region have been synthesized. The developed IR dyes were studied using a biochip-based test system for detection of mutations in the BRCA1/BRCA2 and CHECK2 genes associated with breast cancer. The fluorescent label was introduced to the analyzed DNA during PCR using primers labeled with the synthesized IR dyes. An analyzer that allows recording and processing of images of fluorescent microarrays in the IR spectral region was designed and manufactured. It has been shown that the use of the synthesized dyes enables to conduct analysis in the IR region and improve the reliability of medical diagnostic tests due to low fluorescence intensity of sample components as well as of a biochip substrate and the reagents used for analysis.

[Age-related characteristics of the efficacy of different glucocorticosteroids in the therapy of acute lymphoblastic leukemia].

AIM: To determine predictors for decision-making on a differential approach to choosing glucocorticosteroids (GCS) for children and adolescents with acute lymphoblastic leukemia (ALL). SUBJECTS AND METHODS: The analysis covered 1064 primary patients aged to 1 to 18 years with ALL who had been registered at the clinics of Russia and Belorussia in April 2002 to November 2006. Before induction therapy, the patients were randomized into a dexamethasone (DEXA) 6 mg/m2 group (n=539) and a methylprednisolone (MePRED) 60 mg/m2 one (n=525). RESULTS: The entire group showed no statistically significant differences in survival rates between the patients receiving DEXA or MePRED. However, an analysis of age groups revealed the benefits of DEXA in children younger than 14 years (the event-free survival (EFS) was 76±2 and 71±2%, respectively (p=0.048); the overall survival (OS) was 81±2 and 77±2%, respectively (p=0.046); therapy-induced mortality was 6.4% (DEXA) andl 1.1% (MePRED) (p=0.01 4); the rate of isolated extramedullary relapses was 1.5% (DEXA) and 4.4% (MePRED) (p=0.009). At the same time, EFS and OS in 14-to-18-year-old adolescents were statistically significantly higher than in those who used MePRED (EFS, 65±6 and 52±6%, respectively (p=0.087); OS, 72±6 and 61±6%, respectively; (p=0.l 7). CONCLUSION: The findings suggest that it is possible that the choice of a GCS for ALL therapy must be also based on a patient's age. There is a need for further studies of this matter in prospective randomized multicenter trials in children and adolescents.