[The incidence of AZF deletions, CFTR mutations and long alleles of the ar CAG repeats during the primary laboratory diagnostics in a heterogeneous group of infertily men].

AIM: microdeletions in the AZF region of Y-chromosome, compound heterozygotes of severe and mild CFTR mutations, and long CAG-repeats in the androgen receptor gene (AR) as marker of predisposition are frequently studied as genetic causes of male infertility. A simultaneously testing of the panel including biochemical, immunological, cyto- and molecular genetic markers is often performed during the complex laboratory diagnostics in infertile men. The aim of our work was to identify molecular genetic alterations, which are advisable for simultaneously testing in a man with currently uncertain form of infertility, to increase the informativeness of laboratory diagnostics. MATERIALS AND METHODS: a retrospective study of 885 infertile men was conducted. AZF deletions were determined by multiplex PCR using 10 STS-markers (sY83, sY84, sY86, sY127, sY134, sY143, sY152, sY157, sY254, sY255) and two control loci SRY and AMEL with detection in polyacrylamide gel. Mutations in the CFTR gene (F508del, CFTRdel2.3(21kb), I507del, 1677delTA, 2143delT, 2184insA, 394delTT, W1282X, G542X, N1303K, R334W and 5T) were detected by PCR and SNaPshot. For determination of length of the AR CAG-repeat a fragment analysis of fluorescently labeled PCR products on the 3500xl capillary sequencer was performed. RESULTS: AZF deletions were detected in 8.2% of cases. The largest number of deletions was found in the AZFc subregion (58.9%), while a frequency of deletion in AZFa, AZFb or combined deletions of two and three subregions was 5.5%, 12.3% and 23.3%, respectively. Heterozygous carriage of severe CFTR mutations was detected in 4.7% patients. The most frequent mutation was F508del (83.3%), followed by CFTRdel21kb (7.1%) and W1282X (4.8%). The frequency of the mild splicing 5T mutation was 5.3%, and its incidence was significantly higher than in the previously published control group (p=0.002). AR genotyping revealed that the most prevailing allele was 21 (CAG) (21.5%). Long alleles with 27 or more CAG-trinucleotides were identified in 7.5% of the tested cases. In addition, 7 CAG heterozygotes with Kleinfelter syndrome were found. CONCLUSION: during primary complex laboratory diagnostics in a heterogeneous group of infertile men, it is advisable to detect AZF deletions and the most frequent CFTR mutations, including F508del, CFTRdel21kb, 1677delTA, 2143delT, W1282X and 5T. The more comprehensive analysis of CFTR mutations is justified only in patients with verified obstructive infertility. Sequencing of panels associated with infertility genes using NGS technology is promising.

Structural Alterations in Human Fibroblast Growth Factor Receptors in Carcinogenesis.

Fibroblast growth factor (FGF) plays an important role in human embryogenesis, angiogenesis, cell proliferation, and differentiation. Carcinogenesis is accompanied by aberrant constitutive activation of FGF receptors (FGFRs) resulting from missense mutation in the FGFR1-4 genes, generation of chimeric oncogenes, FGFR1-4 gene amplification, alternative splicing shift toward formation of mesenchymal FGFR isoforms, and FGFR overexpression. Altogether, these alterations contribute to auto- and paracrine stimulation of cancer cells and neoangiogenesis. Certain missense mutations are found at a high rate in urinary bladder cancer and can be used for non-invasive cancer recurrence diagnostics by analyzing urine cell pellet DNA. Chimeric FGFR1/3 and amplified FGFR1/2 genes can predict cell response to the targeted therapy in various oncological diseases. In recent years, high-throughput sequencing has been used to analyze exomes of virtually all human tumors, which allowed to construct phylogenetic trees of clonal cancer evolution with special emphasis on driver mutations in FGFR1-4 genes. At present, FGFR blockers, such as multi-kinase inhibitors, specific FGFR inhibitors, and FGF ligand traps are being tested in clinical trials. In this review, we discuss current data on the functioning of the FGFR family proteins in both normal and cancer cells, mutations in the FGFR1-4 genes, and mechanisms underlying their oncogenic potential, which might be interesting to a broad range of scientists searching for specific tumor markers and targeted anti-cancer drugs.

[Transcription Factor SAP30 Is Involved in the Activation of NETO2 Gene Expression in Clear Cell Renal Cell Carcinoma].

Clear cell renal cell carcinoma (ccRCC) is a common oncourological disease with a high mortality level. The incidence of this type of cancer is constantly increasing, while molecular mechanisms involved in the disease initiation and progression remain far from being fully understood. A problem of the search for novel markers is crucial for improvement of diagnosis and therapy of ccRCC. We have previously found that the disease is characterized by increased expression of the NETO2 gene. In the present study, we showed that isoform 1 (NM_018092.4) makes the main contribution to the upregulation of this gene. Using original CrossHub software, "The Cancer Genome Atlas" (TCGA) project data were analyzed to identify possible mechanisms of NETO2 gene activation in ccRCC. The absence of significant contribution of methylation to the increase of mRNA level of the gene was observed. At the same time, a number of genes encoding transcription factors, which could potentially regulate the expression of NETO2 in ccRCC, were identified. Three such genes (MYCBP, JMY, and SAP30) were selected for the further analysis of their mRNA levels in a set of ccRCC samples with quantitative PCR. We showed a significant increase in mRNA level of one of the examined genes, SAP30, and revealed its positive correlation with NETO2 gene expression. Thus, upregulation of NETO2 gene is first stipulated by the isoform 1 (NM_018092.4), and the probable mechanism of its activation is associated with the increased expression of SAP30 transcription factor.

[Suppression of NR0B2 gene in Clear Cell Renal Cell Carcinoma Is Associated with Hypermethylation of Its Promoter].

Clear cell renal cell carcinoma (ccRCC) is a common urologic malignancy. Understanding of the transcriptional regulation of oncogenes and tumor suppressor genes involved is critical for the development of the treatments for renal tumors. Using ccRCC subdivision of the TCGA dataset, we identified NR0B2 encoding orphan nuclear receptor as a tumor suppressor candidate in renal tissue. In independent cohort of primary renal tumors, quantitative PCR experiments confirmed significant suppression of NR0B2 mRNA in 86% of ccRCC samples studied. In 80% of these cases, we detected the hypermethylation of the NR0B2 pro-moter region. These results suggest that NR0B2 is a tumor suppressor gene in ccRCC, and that the hypermethylation of promoter region is the main mechanism of its downregulation.

[Overexpression of microRNAs miR-9, -98, and -199 Correlates with the Downregulation of HK2 Expression in Colorectal Cancer].

Glycolysis activation is one of the main features of energy metabolism in cancer cells that is associated with the increase in glycolytic enzyme synthesis, primarily, hexokinases (HKs), in many types of tumors. Conversely, in colorectal cancer (CRC) the decrease in the expression of HK2 gene, which encodes one of the key rate-limiting enzyme of glycolysis, was revealed, thus, the study of the mechanisms of its inhibition in CRC is of particular interest. To search for potential microRNAs, inhibiting the expression of HK2 in CRC, we have performed the analysis of data from "The Cancer Genome Atlas" (TCGA) and five microRNA-mRNA target interaction databases (TargetScan, DIANA microT, mirSVR (miRanda), PicTar, and miRTarBase) using original CrossHub software. Seven microRNAs containing binding site on mRNA HK2, which expression is negatively correlated with HK2 expression, were selected for further analysis. The expression levels of these microRNAs and mRNA HK2 were estimated by quantitative PCR on a set of CRC samples. It has been shown, that the expression of three microRNAs (miR-9-5p, -98-5p, and -199-5p) was increased and correlated negatively with mRNA level of HK2 gene. Thus, downregulation of HK2 gene may be caused by its negative regulation through microRNAs miR-9-5p, -98-5p, and -199-5p.

[Biomarkers of prostate cancer sensitivity to the Sendai virus].

Metastatic prostate cancer is often associated with either primary or intractable castration-resistant prostate cancer (CRPC), thus justifying the search for entirely new ways of treatment. Oncolytic viruses are able to selectively induce the death of tumor cells without affecting normal cells. A murine Sendai virus has potential to be used as an oncolytic agent. However, tumors vary in their sensitivity to different viruses, prompting us to attempt to identify corresponding biomarkers that reflect the interaction of cancer cells and the virus. Here, we show that the sensitivity of primary prostatic adenocarcinoma cell lines to Sendai virus strain (SeVM) vary substantially. Using quantitative PCR, we evaluated expression levels of genes that encode RIG-1-like and Toll-like receptors (TLRs) in cell lines and showed that the levels of mRNAs that encode TLR3 and TLR7 correlate with a degree of sensitivity of the cells to Sendai virus. The lines with lower levels of TLR3 and TLR7 expression are more sensitive to the virus.

5-Aminolevulinic acid in intraoperative photodynamic therapy of bladder cancer (results of multicenter trial).

The results of multicenter prospective trial for efficacy of combined modality treatment: transurethral resection (TUR)+photodynamic therapy (PDT) with alasens for bladder cancer are represented in the article. Trials were organized by Research Institute of Organic Intermediates and Dyes and conducted according to clinical protocol approved by Ministry of Health of Russia, at the sites of leading Russian cancer clinical centers. The trial included 45 subjects with verified diagnosis of non-muscle-invasive bladder cancer. Patients underwent TUR of bladder with simultaneous PDT as anti-relapse treatment. Alasens was administered to patients as intravesicular instillation of 3% solution in volume of 50ml with 1.5-2h exposure (prior to TUR). TUR was performed after instillation. PDT session was conducted immediately after the completion of TUR on a single occasion by means of combined local irradiation on tumor bed with diffuse irradiation on whole urinary bladder mucosa (light dose of local irradiation - 100J/cm2, diffuse irradiation - 20J/cm2). Good tolerance of the treatment was noticed, there were no complications. Among 45 patients included in the trial, 35 (78%) completed 12 month protocol follow-up without relapse. In our study PDT with alasens after TUR reported a recurrence rate of non-muscle-invasive bladder cancer for 1st year after treatment of 22%. TUR with intraoperative PDT with 5-aminolevulinic acid may offer an alternative in the treatment of non-muscle-invasive intermediate and high-risk bladder cancer.

[Molecular genetic mechanisms of drug resistance in prostate cancer].

The major problem in prostate cancer treatment is the development of drug resistance and especially important, cross-resistance. The mechanisms of drug resistance, which are divided into ligand-dependent (requiring the presence of androgens in the cell) and independent (not requiring the presence of androgens) are reviewed. The mechanisms are mainly represented with mutations of the androgen receptor and expression of aberrant constitutively active splice variants, as well as up-regulation of genes involved in androgens synthesis.