Transcriptional Activity of Some Genes Involved in Apoptosis in Patients with Vulvar Lichen Sclerosus.

Lichen sclerosus of the vulva is a common, but poorly studied disease. We assessed the level of transcriptional activity of APAF1, BAX, BCL2, BIRC5, CCND1, DAPK1, MCL1, and MYC genes encoding products that control apoptosis in the samples of tissues affected by vulvar lichen sclerosus and adjacent control tissues (n=24). Analysis of transcriptional activity was performed by real-time PCR using specific primers and SYBR Green intercalating dye. After the total group was divided by the presence of the concomitant gynecological diseases, a significant increase in the transcriptional activity of the CCND1 gene was revealed in patients with concomitant uterine fibroids. This may indicate the possible role of the activation of mitosis during tumor initiation.

[A clinical and genetic study of impulsive-compulsive disorders in patients with Parkinson's disease]. / Kliniko-geneticheskoe issledovanie impul'sivno-kompul'sivnykh rasstroistv u bol'nykh bolezn'iu Parkinsona.

AIM: To evaluate the frequency and spectrum of impulsive-compulsive disorders (ICDs) in patients with Parkinson's disease (PD) without dopaminergic medication and among patients receiving dopaminergic replacement therapy, depending on personality type, genetic factors, and to determine the influence of ICDs on the development of other non-motor manifestations of the disease. MATERIAL AND METHODS: Three hundred and eighty-six consecutive patients with idiopathic PD, including untreated patients with PD (de novo) and patients receiving dopaminergic replacement therapy during one year, were examined. ICDs were evaluated with QUIP and diagnostic criteria. Personality type and temperament features were assessed by the Eysenck personality inventory. Genotyping for the single nucleotide polymorphism rs141116007 in the DBH gene involved in the pathogenesis of PD and ICDs was performed. RESULTS AND CONCLUSION: ICDs were identified in 20.2% patients with PD and in 4% patients of the de novo group. The most common (10.36%) behavioral disorder was a binge eating. The frequency of ICDs among patients with PD before the onset of dopamine replacement therapy increased by 1.03 times after one year treatment. Smoking and young age were risk factors for ICDs (p<0.05). The results of the study allowed the determination of social and neuropsychological risk factors for ICDs in patients with PD. The account of these features, as well as early detection of ICDs using screening questionnaires may help to personalize treatment of patients with PD and to prevent the risk of developing comorbid non-motor manifestations of the disease.

[Methylation of the genes for the microRNAs miR-129-2 and miR-9-1, changes in their expression, and activation of their potential target genes in clear cell renal cell carcinoma].

Methylation of promoter CpG islands and microRNA (miRNA) interactions with mRNAs of target genes are epigenetic mechanisms that play a crucial role in deregulation of gene expression and signaling pathways in tumors. Altered expression of six chromosome 3p genes (RARB(2), SEMA3B, RHOA, GPX1, NKIRAS1, and CHL1) and two miRNA genes (MIR-129-2 and MIR-9-1) was observed in primary clear cell renal cell carcinomas (ccRCCs, 31-48 samples) by RT-PCR and qPCR. Significant downregulation (p < 0.05, Fisher's exact test) was observed for SEMA3B, NKIRAS1, and CHL1; and differential expression, for the other chromosome 3p and miRNA genes. Methylation-specific PCR with primers to RARB(2), SEMA3B, MIR-129-2, and MIR-9-1 showed that their methylation frequency was significantly (p < 0.05, Fisher's exact test) elevated in the ccRCC samples. Significant correlations between promoter methylation and expression were confirmed for SEMA3B and observed for the first time for RARB(2), GPX1, and MIR-129-2 in ccRCC (Spearman's correlation coefficient rs ranging 0.31-0.60, p < 0.05). The MIR-129-2 and RARB(2) methylation frequencies significantly correlated with ccRCC progression. MIR-129-2 methylation correlated with upregulation of RARB(2), RHOA, NKIRAS1, and CHL1 (rs ranging 0.35-0.53, p < 0.05). The findings implicate methylation in regulating RARB(2), SEMA3B, GPX1, and MIR-129-2 and indicate that miR-129-2 and methylation of its gene affect RARB(2), RHOA, NKIRAS1, and CHL1 expression.

[Migraine and depression: efficacy and safety of antidepressant therapy]. / Effektivnost' i bezopasnost' terapii antidepressantami migreni i depressii.

AIM: To study an influence of depression on the course of migraine and the efficacy of antidepressants in treatment of depression associated with migraine. MATERIAL AND METHODS: The study consisted of 544 patients with migraine including 240 patients with depression. Patients were examined at the first visit, after 6 months (visit 2) and one year (visit 3). Then patients were interviewed by phone once a year (visits 5, 6, 7, 8), they were asked about a number of days with migraine. Patients with depression were treated with antidepressants. Results and сonclusion. Six types of migraine course were determined: persistent episodic migraine, migraine remission, episodic migraine chronification, chronic migraine regress to episodic migraine, persistent chronic migraine, remitted or wavelike chronic migraine. The presence of depression and skin allodynia predicted the development of persistent and remitted chronic migraine. The effect of antidepressants of different groups was noted in treatment of patients with the combination of chronic migraine and depression. The most pronounced effect was observed with tricyclic antidepressants, the smallest one when selective serotonin reuptake inhibitors were used.

RHOA, SEMA3B, and CKAP2 expression in leukaemia of different types: the results of a pilot experiment.

The transcriptional activity of RHOA, SEMA3B, and CKAP2 genes was assessed in blood samples of leukaemia patients and healthy donors. In the blood of healthy donors, RHOA and CKAP2 gene expression was not detected, and low SEMA3B gene expression was observed. Significant elevation of expression of all the three genes was shown in the case of acute myelogenous leukaemia. In cases of remission of acute lymphoblastic leukaemia and myelodysplastic syndrome, no expression of all three genes was detected. The long isoform of the CKAP2 gene was highly expressed in most analysed types of leukaemia.

[Brain-derived neurotrophic factor gene (BDNF) polymorphism among Moscow citizens].

Recent studies showed that brain-derived neurotrophic factor (BDNF) can participate in pathogenesis of various CNS disorders, being connected with proliferation, differentiation, and survival of neurons. In present study, analysis of occurrence rate was performed for three single nucleotide polymorphisms (SNPs) located in BDNF gene (rs6267 (A/G) allele A-0.265; rs2049046 (A/T) allele A-0.407; rs11030107 (A/G) allele A-0.872) in randomized selection of Moscow citizens. Linkage disequilibrium of rs6165 and rs2049046 loci was shown. Differences in allele frequencies in studied selection and populations of other re- gions were discovered.

[Alteration of SEMA3B gene expression levels in epithelial tumors].

Gene expression decreasing in tumors permits to suggest tumor-suppressor activities for these genes. Thus, mRNA quantity decrease was found for SEMA3B gene in many cell lines of small cell (SCLC) and non-small cell lung carcinoma (NSCLC) and it is well-known that SEMA3B suppresses growth of the NCI-H1299 non-small cell lung carcinoma (NSCLC) cell line and tumor formation in nude mice. The aim of this work was to study spectrum of SEMA3B expression level in epithelial tumors of various locations. Using semi-quantitative RT-PCR it was shown for the first time decrease of SEMA3B mRNA quantity (10-250 times as much) in cell lines of renal, breast and ovarian tumors (4/11, 36%). SEMA3B expression profiles in primary tumors of five locations (kidney, lung, breast, ovary and colon) were studied for the first time. This analysis revealed decrease of mRNA quantity (5-1000 times as much) in clear cell renal cell carcinomas with significant high frequency: 25/51, 49% (cases with decrease of mRNA quantity) and 5/51, 10% (cases with increase), P < 0.0001 by Fisher exact test. In addition, the first data about comparatively frequent decrease of mRNA quantity in ovarian (5/16, 31% vs. 2/16, 12%) and colorectal carcinomas (6/11, 54% vs. 2/11,18%) were shown. These results permitted to suggest a possible role of SEMA3B in inhibiting of growth of renal, ovarian and colorectal cancer cells.

[A new member of the WAS genes family (WASF4)].

The exon-intron structure of the human WASF4 gene has been determined. The in silico analysis of the gene promoter region was performed and the presence of transcription factor binding sites was shown. The highest similarity between the WASF4 protein and the human WASF2 protein was revealed. The WASF4 gene homolog was found in chimpanzee and macaque genomes; WASF4 like nucleotide sequences were not found in other vertebrate genomes. The WASF4 gene expression in human tissues was not detected.

[Activation of RHOA gene transcription in epithelial tumors may be caused by gene amplification and/or demethylation of its promotor region].

RHOA protein, a member of small GTPases family, is implicated in cell morphogenesis, adhesion, and in cell cycle regulation. RHOA gene (3p21.31) exhibits cell transformation activity, and therefore gene is considered as a potential oncogene. The aim of this study was to investigate RHOA transcription and copy number changes in three epithelial tumors (breast, renal cell and epithelial ovarian carcinomas, 45 tumor/normal pairs altogether). EII, HhaI, AciI n Bsh1236I). Hypomethylation of the RHOA promoter region in tumor DNA was observed two times more frequently than increased methylation. Moreover, all (15) cancer cases with hypomethylation of the RHOA gene showed a 2-10 fold increased expression of RHOA. It was concluded that gene copy multiplication and demethylation of the RHOA promoter region can contribute to transcription activation of this gene in epithelial tumors.

TGFalpha reactivates imprinted Igf2 in the parthenogenetic mice embryos and placenta.

Imprinted genes play important roles in the mammalian development. In the parthenogenetic embryos (PE) there is only expression of maternally expressed genes. Therefore, PEs are appropriate experimental models to study genomic imprinting controlling mechanisms. The maternally expressed H19 and paternally expressed Igf2 are reciprocally imprinted genes in normal embryos. Here we studied effect of transforming growth factor alpha (TGFalpha) treatment in vitro (10 ng/ml at the morula stage) on the expression of Igf2/H19 locus in mice PE (9.5-days of gestation, 25 somites) and their placentas (PP). Using RT-PCR we showed that TGFalpha reactivated maternally imprinted Igf2 gene in parthenogenetic embryos and placentas. In spite of similar Tgfalpha expression in the pre-implantation stages, its expression in the 9.5-day parthenogenetic embryos is significantly less than in normal embryos (NE). In our experiments it was shown that reactivation of Igf2 gene occurred independently of H19 gene. In vitro TGFalpha treatment of mouse PE reactivated paternally expressed Igf2 gene in the PE and PP. In the PE and PP both Igf2 and H19 were expressed. It seems that TGFalpha can play an important role as modulator of the Igf2/H19 locus.

[Comparison CCR5de132 mutation in the CCR5 gene frequencies in Russians, Tuvinians, and in different groups of HIV-infected individuals].

The 32-bp deletion (CCR5del32 mutation) in the CCR5 (chemokine (C-C motif) receptor 5) gene, encoding CCR5 chemokine receptor, is one of the factors determining natural resistance to human immunodeficiency virus (HIV-1) infection. In the present study, the samples of Russians (n = 107), Tuvinians (n = 50), and HIV-infected individuals were examined for the presence of CCR5del32 mutation in the CCR5 gene. The CCR5del32 allele frequency in Russians and Tuvinians constituted 7.84 and 2%, respectively. Among HIV-1 infected individuals, two groups, of macrophage-tropic HIV-1 strain- and T-cell-tropic HIV-1 strain-infected were distinguished. The CCR5del32 allele frequency in the first group (6.45%) was lower than in the second one (8.73%). Statistical treatment of the HIV-1 infected individuals typing data showed that the difference in the CCR5del32 allele frequencies between the groups of sexually (macrophage-tropic) and parenterally (T-cell-tropic) infected individuals observed was within the limit of random deviation.

[NotI-STS markers for human chromosome 3 are gene markers].

Ninety four NotI-STS markers to seventy two individual NotI clones were developed basing on DNA nucleotide sequences from NotI-"jumping" and "linking" NotI-libraries of human chromosome 3. The localization of NotI-STS markers and their ordering on chromosome was established by combined data of RH-mapping (our data), contig-mapping, cytogenetic mapping and in silico mapping. Performed comparison of NotI-STS DNAs with human genome sequences revealed two gaps in the regions, 3p21.33 (marker NLI-256) and 3p21.31 (NL3-005), and segmental duplication. Identical DNA fragments are localized in the regions 12q and 3p22-21.33 (marker NL3-007). In the region 3q28-q29 (marker NLM-084) a fragment was detected with its identical copies present also on chromosomes 1, 2, 15 and 19. For 69 NotI-STSs, significant homologies with nucleotide sequences of 70 genes and two cDNAs were detected taking in consideration homologies to NotI-STS 5'- and 3'-terminal sequences. Association of NotI-STSs with genes is confirmed by high correlation of gene density distribution with the density of NotI-STS markers on the map of human chromosome 3. Obtained data evidence possibility of NotI-STS marker application as gene markers and allow considering constructed NotI-map as gene map of human chromosome 3.

[The structure of the human oncogenesis-associated CKAP2 (LB1) gene]. / Struktura sviazannogo s onkogenezom gena CKAP2 (LB1) cheloveka.

The structure was described for human CKAP2, which codes for the cytoskeleton-associated protein 2, is at the boundary of chromosome regions 13q14.3 and 13q21.1, and is rearranged in various tumors. The CKAP2 exonintron structure was established by collating the nucleotide sequences of the cDNA and genomic clone AL359513 (GenBank) and analyzing the gene sequence with the GENSCAN program. The results were verified by amplifying gene fragments. CKAP2 comprises nine exons ranging 70-1442 bp and is about 22 kb in size (regulatory regions included). The CKAP2 promoter contains CCAAT (-39...-33) rather than the canonical TATA box, and harbors nine binding sites for six transcription factors. Thus, CKAP2 possesses all structural elements characteristic of eukaryotic genes. The results may be used to study the CKAP2 expression in normal and tumor cells in order to elucidate its role in carcinogenesis.

Human chromosome 3: integration of 60 NotI clones into a physical and gene map.

Sequence tagged sites generated for 60 NotI clones (NotI-STSs) from human chromosome 3-specific NotI-jumping and NotI-linking libraries were physically located using PCR screening of a radiation hybrid (RH) GeneBridge4 panel. The NotI map of chromosome 3 was generated using these RH-mapping data and those obtained earlier by FISH and sequencing of the corresponding NotI clones. The sequences of the NotI clones showed significant homologies with known genes and/or ESTs for 58 NotI-STSs (97%). These 58 NotI clones displayed 91-100% identity to 54 genes and 23 cDNA/EST clones. One known and two hypothetical protein-coding genes were localized for the first time and nine cDNA clones (unknown genes) were also carefully mapped only in this work. Three newly mapped genes are histone gene H1X (NR1-BK20C) and genes for hypothetical proteins THC1032178 and THC1024604 (NL1-243).

[Brain-specific sequence Hfb1 is part of the 3'-untranslated region of the human complexin 2 gene: new data from in vitro and in silico experiments]. / Mozgospetsificheskaia posledovatel'nost' Hfb1 vkhodit v sostav protiazhennoi 3'-netransliruemoi oblasti gena komplksina 2 cheloveka: novye dannye in vitro i in silico éksperimentov.

A genomic clone hybridizing with brain-specific sequence Hfb1 was isolated from a chromosome 5 consmid library. Hfb1 proved to correspond to a new gene exon which codes for a large 3'-untranslated region of the mRNA for synaptic protein complexin 2. Together with the 985-nt Hfb1 cDNA (EMBL Y15167) isolated previously from a cDNA library of the frontal cerebral cortex, the primary structure was established for genomic clone Ghfb sized more than 4 kb. A GenBank search revealed complete identity of the 5' end of Ghfb and the 3'-untranslated region (878-933) of the human complexin 2 mRNA. Large transcripts with the 5' end corresponding to the complexin 2 mRNA and the 3' end to Ghfb were detected in total mRNA of the human brain by means of RT-PCR. The size of the 3'-untranslated region of the human complexin 2 mRNA was estimated at 4 kb.

[Mapping of EST- and STS-markers in the human genome using a panel fo radiation hybrids]. / Kartirovanie v genome cheloveka EST- i STS-markerov s ispol'zovaniem paneli radiatsionnykh gibridov.

Ten DNA markers were localized in the human genome by a screening procedure against the radiation hybrid somatic cell panel (GeneBridge 4 RH Panel) using polymerase chain reaction (RH mapping method). DNA markers were developed to nucleotide sequences adjacent to NotI sites of human chromosome 3 (NotI-STS markers) and also to nucleotide sequences of human cDNA (EST markers). Three EST markers mapped (B10164, S16R and 18F5R) were localized in the human genome for the first time. Marker B10164 was found to be homologous to the nucleotide sequence of the BASP1 gene coding a major receptor protein. Markers S16R and 18F5R presumably tagged new genes, because no homologies were revealed among the nucleotide sequences presented in the databases. For four NotI-STS, more precise localization on human chromosome 3 was determined. On the basis of the data obtained, the NotI map may be integrated with other types of physical maps of human chromosome 3. RH mapping with a standard commercial panel of radiation hybrid somatic cells provided a chance to integrate the data obtained into international databases and existing integrated human chromosomal maps.

[Radiation mapping as one of main methods of creating high resolution maps of human and animal genomes]. / Radiatsionnoe kartirovanie kak odin iz osnovnykh metodov postroeniia fizicheskikh kart genomov cheloveka i zhivotnykh s vysokim urovnem razresheniia.

Radiation hybrid mapping (RH mapping) is considered as one of the main methods of constructing physical maps of mammalian genomes. In introduction, theoretical prerequisites of developing of the RH mapping and statistical methods of data analysis are discussed. Comparative characteristics of universal commercial panels of the radiation hybrid somatic cells (RH panels) are shown. In experimental part of the work, RH mapping is used to localise nucleotide sequences adjacent to NotI sites of human chromosome 3 with the aim to integrate contig map of NotI clones to comprehensive maps of human genome. Five nucleotide sequences adjacent to the sites of integration of papilloma virus in human genome and expressed in the cells of cervical cancer were localised. It was demonstrated that the region 13q14.3-q21.1 was enriched with nucleotide sequences involved in the processes of oncogenesis. RH mapping can be considered as one of the most perspective applications of the modern radiation biology in the field of molecular genetics, that is, in constructing physical maps of mammalian genomes with high resolution level.