Eugene Klimov

Analysis of a new homozygous deletion in the tumor suppressor region at 3p12.3 reveals two novel intronic noncoding RNA genes

Homozygous deletions or loss of heterozygosity (LOH) at human chromosome band 3p12 are consistent features of lung and other malignancies, suggesting the presence of a tumor suppressor gene(s) (TSG) at this location. Only one gene has been cloned thus far from the overlapping region deleted in lung and breast cancer cell lines U2020, NCI H2198, and HCC38. It is DUTT1 (Deleted in U Twenty Twenty), also known as ROBO1, FLJ21882, and SAX3, according to HUGO. DUTT1, the human ortholog of the fly gene ROBO, has homology with NCAM proteins. Extensive analyses of DUTT1 in lung cancer have not revealed any mutations, suggesting that another gene(s) at this location could be of importance in lung cancer initiation and progression. Here, we report the discovery of a new, small, homozygous deletion in the small cell lung cancer (SCLC) cell line GLC20, nested in the overlapping, critical region. The deletion was delineated using several polymorphic markers and three overlapping P1 phage clones. Fiber‐FISH experiments revealed the deletion was approximately 130 kb. Comparative genomic sequence analysis uncovered short sequence elements highly conserved among mammalian genomes and the chicken genome. The discovery of two EST clusters within the deleted region led to the isolation of two noncoding RNA (ncRNA) genes. These were subsequently found differentially expressed in various tumors when compared to their normal tissues. The ncRNA and other highly conserved sequence elements in the deleted region may represent miRNA targets of importance in cancer initiation or progression. Published 2006 Wiley‐Liss, Inc.

Human papilloma viruses and cervical tumours: mapping of integration sites and analysis of adjacent cellular sequences

BackgroundIn cervical tumours the integration of human papilloma viruses (HPV) transcripts often results in the generation of transcripts that consist of hybrids of viral and cellular sequences. Mapping data using a variety of techniques has demonstrated that HPV integration occurred without obvious specificity into human genome. However, these techniques could not demonstrate whether integration resulted in the generation of transcripts encoding viral or viral-cellular sequences. The aim of this work was to map the integration sites of HPV DNA and to analyse the adjacent cellular sequences.MethodsAmplification of the INTs was done by the APOT technique. The APOT products were sequenced according to standard protocols. The analysis of the sequences was performed using BLASTN program and public databases. To localise the INTs PCR-based screening of GeneBridge4-RH-panel was used.ResultsTwelve cellular sequences adjacent to integrated HPV16 (INT markers) expressed in squamous cell cervical carcinomas were isolated. For 11 INT markers homologous human genomic sequences were readily identified and 9 of these showed significant homologies to known genes/ESTs. Using the known locations of homologous cDNAs and the RH-mapping techniques, mapping studies showed that the INTs are distributed among different human chromosomes for each tumour sample and are located in regions with the high levels of expression.ConclusionsIntegration of HPV genomes occurs into the different human chromosomes but into regions that contain highly transcribed genes. One interpretation of these studies is that integration of HPV occurs into decondensed regions, which are more accessible for integration of foreign DNA.

Altered Expression of the SEMA3B gene in Epithelial Tumors

Tumor-specific expression downregulation may be indicative of a gene’s involvement in tumor suppression. For instance, SEMA3B mRNA levels are decreased in many cell lines of small-cell and non-small cell lung cancer, and SEMA3B was shown to suppress the growth of the NSCLC cell line NCI-H1299 and tumor formation in immunodeficient mice. In this work, SEMA3B expression levels were determined in epithelial tumors of different localizations. In cell lines of renal, breast, and ovarian cancer, SEMA3B mRNA levels were frequently (4/11, 36%) decreased as much as 10–250-fold according to semiquantitative RT-PCR assay. SEMA3B expression levels were also determined in primary tumor extracts of kidney, lung, breast, ovarian, and colorectal cancer. In clear cell renal cell carcinoma, SEMA3B expression was decreased 5–1000-fold in 25 of 51 extracts (49%) compared to 5/51 (10%) extracts with increased mRNA levels; the result was highly significant: P < 0.0001 by Fisher’s exact test. SEMA3B was frequently downregulated in ovarian (5/16, 31% vs. 2/16, 12%) and colorectal cancer (6/11, 54% vs. 2/11, 18%). These results suggest that SEMA3B is involved in the suppression of kidney, ovarian, and colon tumor growth.

Activation of RHOA transcription in epithelial tumors may be caused by gene amplification and/or demethylation of the promoter region

RHOA is a small GTPase involved in morphogenesis, cell adhesion, and cell cycle control. RHOA (3p21.31) is a potential oncogene, causing cell malignant transformation. A study was made of the mechanisms activating RHOA transcription in several epithelial tumors. Semiquantitative RT-PCR revealed elevated transcription of RHOA in tumors (45 cases of breast cancer, renal cell carcinoma, and epithelial ovarian carcinoma; p < 10−4). For the first time transcriptional activation of RHOA in tumors was reliably associated with its amplification (p = 10−7). Digestion with four methyl-sensitive restriction enzymes (HpaII, HhaI, AciI, and Bsh1236I) and subsequent PCR revealed a change in methylation of the RHOA promoter region in 23 out of 45 tumors, hypomethylation being two times more frequent than hypermethylation. Demethylation of the RHOA promoter region was associated with a two-to tenfold increase in the transcription level of the gene. Thus, two mechanisms—amplification and demethylation of the promoter region—were shown to change the level of RHOA transcription in epithelial tumors.

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).

Effects of MTHFR gene polymorphism on the clinical and electrophysiological characteristics of migraine

BackgroundIt was previously shown that the MTHFR gene polymorphism correlated with an increased risk of migraine, particularly migraine with aura. The substitution of cytosine for thymine at the position 677 of the MTHFR gene leads to formation of the thermolabile form of the protein and development of hyperhomocysteinemia, which increases the probability of migraine. The purpose of this study was to determine whether the replacement of C677T in the gene MTHFR influenced any particular symptoms of the disease.MethodsWe have analyzed clinical and electrophysiological characteristics of 83 patients with migraine (migraine with aura (MA), 19 patients, and migraine without aura (MO), 64 patients, according to the ICHD-II (2003)) taking into account their genotypes of C677T variant of MTHFR.ResultsWe have shown that MA was significantly more prevalent among the T-allele carriers (37.2%), as compared to the СС genotype patients (0%), р < 0.0001. Patients with TT genotype were not only more likely to have accompanying symptoms (significant differences were found only for photophobia), but also more sensitive to migraine attack triggers. In RP-VEP test results we observed a trend that the T-allele carriers were presented with the decreased N75/P100 amplitudes and a positive habituation index, as compared to the СС genotype patients.ConclusionsThus, according to our data, the MTHFR genotypes are associated with several clinical and electrophysiological characteristics of migraine.

NotI Sequence-Tagged Sites as Markers of Genes on Human Chromosome 3

Using nucleotide sequences from jumping and linking NotI libraries of human chromosome 3, 94 NotI-STS markers for 72 individual NotI clones were developed. The positions of the NotI-STS markers and their order on the chromosome were determined by a combination of RH-mapping (our data), contig mapping, cytogenetic mapping, and in silico mapping. Comparison of NotI-STS DNAs with human genome sequences revealed two gaps in the regions 3p21.33 (marker NL1-256) and 3p21.31 (NL3-005), and a segmental duplication. Identical DNA fragments were found in the regions 12q and 3p22–21.33 (marker NL3-007). In the 3q28–q29 region (marker NLM-084), a fragment was detected whose identical copies were also present on chromosomes 1, 2, 15, and 19. For 69 NotI-STSs, significant homologies to nucleotide sequences of 70 genes and 2 cDNAs were detected (with homologies in NotI-STS 5′- and 3′-terminal sequences being taken into account). An association between NotI-STSs and genes is confirmed by a strong correlation between the density distributions of genes and NotI-STS markers on the map of human chromosome 3. Our results indicate that the NotI map may be regarded as a gene map of human chromosome 3. Thus, NotI-STSs are applicable as gene markers.

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.

TGFα 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 (TGFα) 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 TGFα reactivated maternally imprinted Igf2 gene in parthenogenetic embryos and placentas. In spite of similar Tgfα expression in the preimplantation 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 TGFα 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 TGFα can play an important role as modulator of the Igf2/H19 locus.

Syndrome of transient headache and neurological deficits with cerebrospinal fluid lymphocytosis (HaNDL)

minutes later, and after another 15 minutes the patient experienced an intense pulsating headache in the right temporal region scoring 8 on the Visual Analogue Scale (VAS) and accompanied by vomiting, photo-, phono-, and osmophobia. The pain persisted for 6-8 hours, and afterwards the patient fell asleep. The patient experienced three more similar episodes with an interval of 2 to 3 days. Two days after the last episode, the patient again woke up with an intense headache, feeling discomfort in an arm and the face. The headache score reached 10 on the VAS. Due to these complaints, the patient was admitted to a hospital.