Identification of a novel prostate cancer susceptibility variant in the KLK3 gene transcript

Abstract Genome-wide association studies (GWAS) have identified more than 30 prostate cancer (PrCa) susceptibility loci. One of these (rs2735839) is located close to a plausible candidate susceptibility gene, KLK3, which encodes prostate-specific antigen (PSA). PSA is widely used as a biomarker for PrCa detection and disease monitoring. To refine the association between PrCa and variants in this region, we used genotyping data from a two-stage GWAS using samples from the UK and Australia, and the Cancer Genetic Markers of Susceptibility (CGEMS) study. Genotypes were imputed for 197 and 312 single nucleotide polymorphisms (SNPs) from HapMap2 and the 1000 Genome Project, respectively. The most significant association with PrCa was with a previously unidentified SNP, rs17632542 (combined P = 3.9 × 10−22). This association was confirmed by direct genotyping in three stages of the UK/Australian GWAS, involving 10,405 cases and 10,681 controls (combined P = 1.9 × 10−34). rs17632542 is also shown to be associated with PSA levels and it is a non-synonymous coding SNP (Ile179Thr) in KLK3. Using molecular dynamic simulation, we showed evidence that this variant has the potential to introduce alterations in the protein or affect RNA splicing. We propose that rs17632542 may directly influence PrCa risk.

Identification of a core promoter and a novel isoform of the human TSCI gene transcript and structural comparison with mouse homolog

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder with loci on chromosome 9q34.12 (TSC1) and chromosome 16p13.3 (TSC2). Genes for both loci have been isolated and characterized. The promoters of both genes have not been characterized so far and little is known about the regulation of these genes. This study reports the characterization of the human TSC1 promoter region for the first time. We have identified a novel alternative isoform in the 5' untranslated region (UTR) of the TSC1 gene transcript involving exon 1. Alternative isoforms in the 5' UTR of the mouse Tsc1 gene transcript involving exon I and exon 2 have also been identified. We have identified three upstream open reading frames (uORFs) in the 5' UTR of the TSC1/Tsc1 gene. A comparative study of the 5' UTR of TSC1/Tsc1 gene has revealed that there is a high degree of similarity not only in the sequence but also in the splicing pattern of both human and mouse TSC1 genes. We have used PCR methodology to isolate approximately 1.6 kb genomic DNA 5' to the TSC1 cDNA. This sequence has directed a high level of expression of luciferase activity in both HeLa and HepG2 cells. Successive 5' and 3' deletion analysis has suggested that a -587 bp region, from position +77 to -510 from the transcription start site (TSS), contains the promoter activity. Interestingly, this region contains no consensus TATA box or CAAT box. However, a 521-bp fragment surrounding the TSS exhibits the characteristics of a CpG island which overlaps with the promoter region. The identification of the TSC1 promoter region will help in designing a suitable strategy to identify mutations in this region in patients who do not show any mutations in the coding regions. It will also help to study the regulation of the TSC1 gene and its role in tumorigenesis. (C) 2003 Elsevier B.V. All rights reserved.

Higher topoisomerase 2 alpha gene transcript levels predict better prognosis in GBM patients receiving temozolomide chemotherapy: identification of temozolomide as a TOP2A inhibitor

The search for molecular markers which predict response to chemotherapy is an important aspect of current neuro-oncology research. MGMT promoter methylation is the only proved marker of glioblastoma. The purpose of this study was to assess the effect of topoisomerase expression on glioblastoma survival and study the mechanisms involved. The transcript levels of all isoforms of the topoisomerase family in all grades of diffuse astrocytoma were assessed. A prospective study of patients with glioblastoma treated by a uniform treatment procedure was performed with the objective of correlating outcome with gene expression. The ability of TOP2A enzyme to relax the super coiled plasmid DNA in the presence of temozolomide was evaluated to assess its effect on TOP2A. The temozolomide cyctotoxicity of TOP2A-silenced U251 cells was assessed. The transcript levels of TOP2A, TOP2B, and TOP3A are upregulated significantly in GBM in comparison with lower grades of astrocytoma and normal brain samples. mRNA levels of TOP2A correlated significantly with survival of the patients. Higher TOP2A transcript levels in GBM patients predicted better prognosis (P = 0.043; HR = 0.889). Interestingly, we noted that temozolomide inhibited TOP2A activity in in-vitro enzyme assays. We also noted that siRNA knock down of TOP2A rendered a glioma cell line resistant to temozolomide chemotherapy. We demonstrated for the first time that temozolomide is also a TOP2A inhibitor and established that TOP2A transcript levels determine the chemosensitivity of glioblastoma to temozolomide therapy. Very high levels of TOP2A are a good prognostic indicator in GBM patients receiving temozolomide chemotherapy.

Sequence confirmation of the EWS-WT1 fusion gene transcript in the peritoneal effusion of a patient with desmoplastic small round cell tumor

Desmoplastic small round cell tumor (DSRCT) is a rare undifferentiated neoplasm. The prognosis is poor, even if therapy is instituted promptly. and thus it is important to differentiate it from other histologically and cytologically similar-looking malignancies of the young adult. We present a case of DSRCT in a 17-yr-old male with disseminated peritoneal disease and peritoneal effusion. The cytology sample showed a malignant small round cell tumor, the classical cytological features of DSRCT, and immunohistochemistry performed in the prepared cell block exhibited an antibody expression profile in keeping with DSRCT. Further material front the effusion was prepared for RNA extraction, following which a reverse-transcriptase polymerase chain reaction (RTPCR) and sequencing of the t(l l;22)(p13;q11 or q12) were carried out. The result showed the presence of the reciprocal translocation and thus confirmed the diagnosis of DSRCT. This case shows how molecular techniques (including sequencing) call be applied to cytology in clarifying and confirming certain difficult diagnosis of undifferentiated neoplasms, DSRCT in this particular case. Diagn. Cytopathol. 2003;29:341-343. (C) 2003 Wiley-Liss. Inc.

Expression Profile and Distribution of Efhc1 Gene Transcript During Rodent Brain Development

One of the putative causative genes for juvenile myoclonic epilepsy (JME) is EFHC1. We report here the expression profile and distribution of Efhc1 messenger RNA (mRNA) during mouse and rat brain development. Real-time polymerase chain reaction revealed that there is no difference in the expression of Efhc1 mRNA between right and left hemispheres in both species. In addition, the highest levels of Efhc1 mRNA were found at intra-uterine stages in mouse and in adulthood in rat. In common, there was a progressive decrease in Efhc1 expression from 1-day-old neonates to 14-day-old animals in both species. In situ hybridization studies showed that rat and mouse Efhc1 mRNAs are expressed in ependymal cells of ventricle walls. Our findings suggest that Efhc1 expression is more important during initial phases of brain development and that at this stage it could be involved in key developmental mechanisms underlying JME.

The TIGR Gene Indices: analysis of gene transcript sequences in highly sampled eukaryotic species

While genome sequencing projects are advancing rapidly, EST sequencing and analysis remains a primary research tool for the identification and categorization of gene sequences in a wide variety of species and an important resource for annotation of genomic sequence. The TIGR Gene Indices (http://www.tigr.org/tdb/tgi.shtml) are a collection of species-specific databases that use a highly refined protocol to analyze EST sequences in an attempt to identify the genes represented by that data and to provide additional information regarding those genes. Gene Indices are constructed by first clustering, then assembling EST and annotated gene sequences from GenBank for the targeted species. This process produces a set of unique, high-fidelity virtual transcripts, or Tentative Consensus (TC) sequences. The TC sequences can be used to provide putative genes with functional annotation, to link the transcripts to mapping and genomic sequence data, to provide links between orthologous and paralogous genes and as a resource for comparative sequence analysis.

Gibberellin Dose-Response Regulation of GA4 Gene Transcript Levels in Arabidopsis1

The gibberellins (GAs) are a complex family of diterpenoid compounds, some of which are potent endogenous regulators of plant growth. As part of a feedback control of endogenous GA levels, active GAs negatively regulate the abundance of mRNA transcripts encoding GA biosynthesis enzymes. For example, Arabidopsis GA4 gene transcripts encode GA 3β-hydroxylase, an enzyme that catalyzes the conversion of inactive to active GAs. Here we show that active GAs regulate GA4 transcript abundance in a dose-dependent manner, and that down-regulation of GA4 transcript abundance is effected by GA4 (the product of 3β-hydroxylation) but not by its immediate precursor GA9 (the substrate). Comparison of several different GA structures showed that GAs active in promoting hypocotyl elongation were also active in regulating GA4 transcript abundance, suggesting that similar GA:receptor and subsequent signal transduction processes control these two responses. It is interesting that these activities were not restricted to 3β-hydroxylated GAs, being also exhibited by structures that were not 3β-hydroxylated but that had another electronegative group at C-3. We also show that GA-mediated control of GA4 transcript abundance is disrupted in the GA-response mutants gai and spy-5. These observations define a sensitive homeostatic mechanism whereby plants may regulate their endogenous GA levels.

Extensive alteration of fungal gene transcript accumulation and elevation of G-protein-regulated cAMP levels by a virulence-attenuating hypovirus.

Persistent infection of the chestnut blight fungus Cryphonectria parasitica with the prototypic hypovirus CHVI-713 results in attenuation of fungal virulence (hypo-virulence) and reduced accumulation of the GTP-binding (G) protein a subunit CPG-1. Transgenic cosuppression of CPG-1 accumulation in the absence of virus infection also confers hypovirulence. We now report the use of mRNA differential display to examine the extent to which virus infection alters fungal gene transcript accumulation and to assess the degree to which modification of CPG-1 signal transduction contributes to this alteration. More than 400 PCR products were identified that either increased (296 products) or decreased (127 products) in abundance as a result of virus infection. Significantly, 65% of these products exhibited similar changes as a result of CPG-1 cosuppression in the absence of virus infection. We also report that both virus infection and CPG-1 cosuppression elevate cAMP levels 3- to 5-fold. Additionally, it was possible to mimic the effect of virus infection and CPG-1 cosuppression on transcript accumulation for representative fungal genes by drug-induced elevation of cAMP levels. These results strengthen and extend previous indications that hypovirus infection causes a significant and persistent alteration of fungal gene expression/transcript accumulation. They further show that this alteration is primarily mediated through modification of the CPG-1 signaling pathway and suggest that, similar to mammalian Gi alpha subunits, CPG-1 functions as a negative modulator of adenylyl cyclase. Finally, these results suggest a role for G-protein-regulated cAMP accumulation in hypovirus-mediated alteration of fungal gene expression.

Gene expression studies in multiple sclerosis

Multiple sclerosis (MS) is a serious neurological disorder affecting young Caucasian individuals, usually with an age of onset at 18 to 40 years old. Females account for approximately 60x of MS cases and the manifestation and course of the disease is highly variable from patient to patient. The disorder is characterised by the development of plaques within the central nervous system (CNS). Many gene expression studies have been undertaken to look at the specific patterns of gene transcript levels in MS. Human tissues and experimental mice were used in these gene-profiling studies and a very valuable and interesting set of data has resulted from these various expression studies. In general, genes showing variable expression include mainly immunological and inflammatory genes, stress and antioxidant genes, as well as metabolic and central nervous system markers. Of particular interest are a number of genes localised to susceptible loci previously shown to be in linkage with MS. However due to the clinical complexity of the disease, the heterogeneity of the tissues used in expression studies, as well as the variable DNA chips/membranes used for the gene profiling, it is difficult to interpret the available information. Although this information is essential for the understanding of the pathogenesis of MS, it is difficult to decipher and define the gene pathways involved in the disorder. Experiments in gene expression profiling in MS have been numerous and lists of candidates are now available for analysis. Researchers have investigated gene expression in peripheral mononuclear white blood cells (PBMCs), in MS animal models Experimental Allergic Encephalomyelitis (EAE) and post mortem MS brain tissues. This review will focus on the results of these studies.

Ethanol exposure alters monoamine oxidase gene-expression in neuronal SH-SY5Y cells

Abstract: Monoamine Oxidase (MAO) enzymes catabolise, and thus modulate abundance of, neurotransmitters in the brain. Variation in MAO enzyme activity has been linked to alcohol abuse behaviour, although the molecular mechanisms underlying this association are not understood. The present study evaluated relative gene-transcript abundance of MAO-A and MAO-B in the SH-SY5Y human neuroblastoma cell-line in response to ethanol exposure and following ethanol withdrawal. We found that each isoform of MAO was significantly transcriptionally up-regulated 55-80% in response to 100mM ethanol exposure. This trend was maintained following prolonged exposures (24 h-72 h) and with short exposures (24 h) followed by a period of ethanol withdrawal, suggesting that the transcriptional regulation is the result of a cellular change occurring within the first 24 hours of ethanol exposure. These results suggest a role for MAO transcriptional regulation in the complex neurobiochemical changes underlying alcohol addiction.

Characterisation of a glutathione reductase gene and its genetic locus from pea (Pisum sativum L.)

A cDNA encoding the chloroplast/mitochondrial form of glutathione reductase (GR:EC 1,6,4,2) from pea (Pisum sativum L.) was used to map a single GR locus, named GORI. In two domesticated genotypes of pea (cv, Birte and JI 399) it is likely that the GORI locus contains a single gene. However, in a semi-domesticated land race of pea sequences were detected but closely related sets of GR gene sequences were in JI 281 represent either a second intact gene or a partial or pseudogene copy. A GR gene was cloned from ev. Birte, sequenced and its structure analysed. No features of the transcription or structure of the gene suggested a mechanism for generating any more than one form of . From these data plus previously published biochemical evidence was suggested a second, distinct gene encoding for the cytosolic form of GR should be present in peas. The GORI-encoded GR mRNA can be detected in all main organs of the plant and no alternative spliced species was present which could perhaps account for the generation of multiple isoforms of GR. The mismatch between the number of charge-separable isoforms in pea and the proposed number suggests that different GR isoforms arise by some form of post-transnational modification.

Age and exposure to arsenic alter base excision repair transcript levels in mice

Arsenic (As) induces DNA-damaging reactive oxygen species. Most oxidative DNA damage is countered by base excision repair (BER), the capacity for which may be reduced in older animals. We examined whether age and consumption of As in lactational milk or drinking water influences BER gene transcript levels in mice. Lactating mothers and 24-week-old mice were exposed (24 h or 2 weeks) to As (2 or 50 p.p.m.) in drinking water. Lung tissue was harvested from adults, neonates (initially 1 week old) feeding from lactating mothers and untreated animals 1– 26 weeks old. Transcripts encoding BER proteins were quantified. BER transcript levels decreased precipitously with age in untreated mice but increased in neonates whose mothers were exposed to 50 p.p.m. As for 24 h or 2 weeks. Treatment of 24-week-old mice with 2 or 50 p.p.m. As for 2 weeks decreased all transcript levels measured. Exposure to As attenuated the age-related transcript level decline for only one BER gene. We conclude that aging is associated with a rapid reduction of BER transcript levels in mice, which may contribute to decreased BER activity in older animals. Levels of As that can alter gene expression are transmitted to neonatal mice in lactational milk produced by mothers drinking water containing As, raising concerns about breastfeeding in countries having As-contaminated groundwater. Reduction of BER transcript levels in 24- week-old mice exposed to As for 2 weeks suggests As may potentiate sensitivity to itself in older animals.

A Gene Coding for Tomato Fruit β-Galactosidase II Is Expressed during Fruit Ripening : Cloning, Characterization, and Expression Pattern

β-Galactosidases (EC 3.2.1.23) constitute a widespread family of enzymes characterized by their ability to hydrolyze terminal, nonreducing β-d-galactosyl residues from β-d-galactosides. Several β-galactosidases, sometimes referred to as exo-galactanases, have been purified from plants and shown to possess in vitro activity against extracted cell wall material via the release of galactose from wall polymers containing β(1→4)-d-galactan. Although β-galactosidase II, a protein present in tomato (Lycopersicon esculentum Mill.) fruit during ripening and capable of degrading tomato fruit galactan, has been purified, cloning of the corresponding gene has been elusive. We report here the cloning of a cDNA, pTomβgal 4 (accession no. AF020390), corresponding to β-galactosidase II, and show that its corresponding gene is expressed during fruit ripening. Northern-blot analysis revealed that the β-galactosidase II gene transcript was detectable at the breaker stage of ripeness, maximum at the turning stage, and present at decreasing levels during the later stages of normal tomato fruit ripening. At the turning stage of ripeness, the transcript was present in all fruit tissues and was highest in the outermost tissues (including the peel). Confirmation that pTomβgal 4 codes for β-galactosidase II was derived from matching protein and deduced amino acid sequences. Furthermore, analysis of the deduced amino acid sequence of pTomβgal 4 suggested a high probability for secretion based on the presence of a hydrophobic leader sequence, a leader-sequence cleavage site, and three possible N-glycosylation sites. The predicted molecular mass and isoelectric point of the pTomβgal 4-encoded mature protein were similar to those reported for the purified β-galactosidase II protein from tomato fruit.

Characterisation of the gene encoding type II DNA topoisomerase from Leishmania donovani: a key molecular target in antileishmanial therapy

The gene encoding type II DNA topoisomerase from the kinetoplastid hemoflagellated protozoan parasite Leishmania donovani (LdTOP2) was isolated from a genomic DNA library of this parasite. DNA sequence analysis revealed an ORF of 3711 bp encoding a putative protein of 1236 amino acids with no introns. The deduced amino acid sequence of LdTOP2 showed strong homologies to TOP2 sequences from other kinetoplastids, namely Crithidia and Trypanosoma spp. with estimated identities of 86 and 68%, respectively. LdTOP2 shares a much lower identity of 32% with its human homologue. LdTOP2 is located as a single copy on a chromosome in the 0.7 Mb region in the L.donovani genome and is expressed as a 5 kb transcript. 5′-Mapping studies indicate that the LdTOP2 gene transcript is matured post-transcriptionally with the trans-splicing of the mini-exon occurring at –639 from the predicted initiation site. Antiserum raised in rabbit against glutathione S-transferase fusion protein containing the major catalytic portion of the recombinant L.donovani topoisomerase II protein could detect a band on western blots at ∼132 kDa, the expected size of the entire protein. Use of the same antiserum for immunolocalisation analysis led to the identification of nuclear, as well as kinetoplast, antigens for L.donovani topoisomerase II. The in vitro biochemical properties of the full-length recombinant LdTOP2 when overexpressed in E.coli were similar to the Mg(II) and ATP-dependent activity found in cell extracts of L.donovani.

High frequency of inactivating mutations in the neurofibromatosis type 2 gene (NF2) in primary malignant mesotheliomas.

Malignant mesotheliomas (MMs) are aggressive tumors that develop most frequently in the pleura of patients exposed to asbestos. In contrast to many other cancers, relatively few molecular alterations have been described in MMs. The most frequent numerical cytogenetic abnormality in MMs is loss of chromosome 22. The neurofibromatosis type 2 gene (NF2) is a tumor suppressor gene assigned to chromosome 22q which plays an important role in the development of familial and spontaneous tumors of neuroectodermal origin. Although MMs have a different histogenic derivation, the frequent abnormalities of chromosome 22 warranted an investigation of the NF2 gene in these tumors. Both cDNAs from 15 MM cell lines and genomic DNAs from 7 matched primary tumors were analyzed for mutations within the NF2 coding region. NF2 mutations predicting either interstitial in-frame deletions or truncation of the NF2-encoded protein (merlin) were detected in eight cell lines (53%), six of which were confirmed in primary tumor DNAs. In two samples that showed NF2 gene transcript alterations, no genomic DNA mutations were detected, suggesting that aberrant splicing may constitute an additional mechanism for merlin inactivation. These findings implicate NF2 in the oncogenesis of primary MMs and provide evidence that this gene can be involved in the development of tumors other than nervous system neoplasms characteristic of the NF2 disorder. In addition, unlike NF2-related tumors, MM derives from the mesoderm; malignancies of this origin have not previously been associated with frequent alterations of the NF2 gene.