Estimation of the diagnostic accuracy of the glyco-C and US9 gene-based polymerase chain reaction technique for the detection of bovine Herpesvirus type 5 DNA in decomposed brain suspension from a slaughter house using Bayesian analysis, Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

COMT Met (158) modulates facial emotion recognition in bipolar I disorder mood episodes

Background: One of the many cognitive deficits reported in bipolar disorder (BD) patients is facial emotion recognition (FER), which has recently been associated with dopaminergic catabolism. Catechol-O-methyltransferase (COMT) is one of the main enzymes involved in the metabolic degradation of dopamine (DA) in the prefrontal cortex (PFC). The COMT gene polymorphism rs4680 (Val(158)Met) Met allele is associated with decreased activity of this enzyme in healthy controls. The objective of this study was to evaluate the influence of Val(158)Met on FER during manic and depressive episodes in BD patients and in healthy controls. Materials and methods: 64 BD type I patients (39 in manic and 25 in depressive episodes) and 75 healthy controls were genotyped for COMT rs4680 and assessed for FER using the Ekman 60 Faces (EK60) and Emotion Hexagon (Hx) tests. Results: Bipolar manic patients carrying the Met allele recognized fewer surprised faces, while depressed patients with the Met allele recognized fewer "angry" and "happy" faces. Healthy homozygous subjects with the Met allele had higher FER scores on the Hx total score, as well as on "disgust" and "angry" faces than other genotypes. Conclusion: This is the first study suggesting that COMT rs4680 modulates FER differently during BD episodes and in healthy controls. This provides evidence that PFC DA is part of the neurobiological mechanisms of social cognition. Further studies on other COMT polymorphisms that include euthymic BD patients are warranted. ClinicalTrials.gov Identifier: NCT00969. (C) 2011 Elsevier B.V. All rights reserved.

Variation in DNA repair gene XRCC3 affects susceptibility to astrocytomas and glioblastomas

The gene XRCC3 (X-ray cross complementing group 3) has the task of repairing damage that occurs when there is recombination between homologous chromosomes. Repair of recombination between homologous chromosomes plays an important role in maintaining genome integrity, although it is known that double-strand breaks are the main inducers of chromosomal aberrations. Changes in the XRCC3 protein lead to an increase in errors in chromosome segregation due to defects in centrosomes, resulting in aneuploidy and other chromosomal aberrations, such as small increases in telomeres. We examined XRCC3 Thr241Met polymorphism using PCR-RFLP in 80 astrocytoma and glioblastoma samples. The individuals of the control group (N = 100) were selected from the general population of the Sao Paulo State. Odds ratio and 95%CI were calculated using a logistic regression model. Patients who had the allele Met of the XRCC3 Thr241Met polymorphism had a significantly increased risk of tumor development (odds ratio = 3.13; 95% confidence interval = 1.50-6.50). There were no significant differences in overall survival of patients. We suggest that XRCC3 Thr241Met polymorphism is involved in susceptibility for developing astrocytomas and glioblastomas.

Correlations of CTLA-4 gene polymorphisms and hepatitis C chronic infection

Background: Cytotoxic T lymphocyte-associated factor 4 (CTLA-4) functions as a negative regulator of T cell-mediated immune response. Molecular changes associated to CTLA-4 gene polymorphisms could reduce its ability to suppress and control lymphocyte proliferation. Aims: To evaluate the frequency of CTLA-4 gene polymorphisms in chronic hepatitis C virus (HCV) infected patients and correlate to clinical and histological findings. Methods: We evaluated 112 HCV-infected subjects prospectively selected and 183 healthy controls. Clinical and liver histological data were analysed. - 318C > T, A49G and CT60 CTLA-4 single-nucleotide polymorphisms (SNPs) were studied by PCR-RFLP and AT(n) polymorphism by DNA fragment analysis by capillary electrophoresis in automatic sequencer. Results: Eight AT repetitions in 3' UTR region were more frequent in HCV-infected subjects. We found a positive association of -318C and + 49G with HCV genotype 3 (P = 0.008, OR 9.13, P = 0.004, OR 2.49 respectively) and an inverse association of both alleles with HCV genotype 1 (P = 0.020, OR 0.19, P = 0.002, OR 0.38 respectively). Allele + 49G was also associated to aminotransferases quotients > 3 (qALT, P = 0.034, qAST, P = 0.041). Allele G of CT60 SNP was also associated with qAST > 3 (P = 0.012). Increased number of AT repetitions was positively associated to severe necroinflammatory activity scores in liver biopsies (P = 0.045, OR 4.62). Conclusion: CTLA-4 gene polymorphisms were associated to HCVinfection. Eight AT repetitions were more prevalent in HCV-infected subjects. - 318C and + 49G alleles were associated to genotypes 1 and 3 infections and increased number of AT repetitions in 3' UTR region favoured severe necroinflammatory activity scores in liver biopsies.

MTP -493G/T gene polymorphism is associated with steatosis in hepatitis C-infected patients

The reduction of hepatic microsomal transfer protein (MTP) activity results in fatty liver, worsening hepatic steatosis and fibrosis in chronic hepatitis C (CHC). The G allele of the MTP gene promoter, -493G/T, has been associated with lower transcriptional activity than the T allele. We investigated this association with metabolic and histological variables in patients with CHC. A total of 174 untreated patients with CHC were genotyped for MTP -493G/T by direct sequencing using PCR. All patients were negative for markers of Wilson’s disease, hemochromatosis and autoimmune diseases and had current and past daily alcohol intake lower than 100 g/week. The sample distribution was in Hardy-Weinberg equilibrium. Among subjects with genotype 1, 56.8% of the patients with fibrosis grade 3+4 presented at least one G allele versus 34.3% of the patients with fibrosis grade 1+2 (OR = 1.8; 95%CI = 1.3-2.3). Logistic regression analysis with steatosis as the dependent variable identified genotypes GG+GT as independent protective factors against steatosis (OR = 0.4, 95%CI = 0.2-0.8; P = 0.01). The results suggest that the presence of the G allele of MTP -493G/T associated with lower hepatic MTP expression protects against steatosis in our CHC patients.

The vitamin D receptor gene bAt (CCA) haplotype impairs the response to pegylated-interferon/ribavirin-based therapy in chronic hepatitis C patients

Chronic hepatitis C infection is a major cause of end-stage liver disease. Therapy outcome is influenced by 25-OH vitamin D deficiency. To further address this observation, our study investigates the impact of the vitamin D receptor (NR1I1) haplotype and combined effects of plasma vitamin D levels in a well-described cohort of hepatitis C patients.

Met-thodology

Met-regulated expression signature defines a subset of human hepatocellular carcinomas with poor prognosis and aggressive phenotype. Kaposi-Novak P, Lee JS, Gomez-Quiroz L, Coulouarn C, Factor VM, Thorgeirsson SS. Identification of specific gene expression signatures characteristic of oncogenic pathways is an important step toward molecular classification of human malignancies. Aberrant activation of the Met signaling pathway is frequently associated with tumour progression and metastasis. In this study, we defined the Met-dependent gene expression signature using global gene expression profiling of WT and Met-deficient primary mouse hepatocytes. Newly identified transcriptional targets of the Met pathway included genes involved in the regulation of oxidative stress responses as well as cell motility, cytoskeletal organization, and angiogenesis. To assess the importance of a Met-regulated gene expression signature, a comparative functional genomic approach was applied to 242 human hepatocellular carcinomas (HCCs) and 7 metastatic liver lesions. Cluster analysis revealed that a subset of human HCCs and all liver metastases shared the Met-induced expression signature. Furthermore, the presence of the Met signature showed significant correlation with increased vascular invasion rate and microvessel density as well as with decreased mean survival time of HCC patients. We conclude that the genetically defined gene expression signatures in combination with comparative functional genomics constitute an attractive paradigm for defining both the function of oncogenic pathways and the clinically relevant subgroups of human cancers. [Abstract reproduced by permission of J Clin Invest 2006;116:1582-1595].

Gene by BMI Interactions Influencing C-Reactive Protein Levels in European-Americans

C-Reactive Protein (CRP) is a biomarker indicating tissue damage, inflammation, and infection. High-sensitivity CRP (hsCRP) is an emerging biomarker often used to estimate an individual’s risk for future coronary heart disease (CHD). hsCRP levels falling below 1.00 mg/l indicate a low risk for developing CHD, levels ranging between 1.00 mg/l and 3.00 mg/l indicate an elevated risk, and levels exceeding 3.00 mg/l indicate high risk. Multiple Genome-Wide Association Studies (GWAS) have identified a number of genetic polymorphisms which influence CRP levels. SNPs implicated in such studies have been found in or near genes of interest including: CRP, APOE, APOC, IL-6, HNF1A, LEPR, and GCKR. A strong positive correlation has also been found to exist between CRP levels and BMI, a known risk factor for CHD and a state of chronic inflammation. We conducted a series of analyses designed to identify loci which interact with BMI to influence CRP levels in a subsample of European-Americans in the ARIC cohort. In a stratified GWA analysis, 15 genetic regions were identified as having significantly (p-value < 2.00*10-3) distinct effects on hsCRP levels between the two obesity strata: lean (18.50 kg/m2 < BMI < 24.99 kg/m2) and obese (BMI ≥ 30.00 kg/m2). A GWA analysis performed on all individuals combined (i.e. not a priori stratified for obesity status) with the inclusion of an additional parameter for BMI by gene interaction, identified 11 regions which interact with BMI to influence hsCRP levels. Two regions containing the genes GJA5 and GJA8 (on chromosome 1) and FBXO11 (on chromosome 2) were identified in both methods of analysis suggesting that these genes possibly interact with BMI to influence hsCRP levels. We speculate that atrial fibrillation (AF), age-related cataracts and the TGF-β pathway may be the biological processes influenced by the interaction of GJA5, GJA8 and FBXO11, respectively, with BMI to cause changes in hsCRP levels. Future studies should focus on the influence of gene x bmi interaction on AF, age-related cataracts and TGF-β.

Requirement for the c-Maf transcription factor in crystallin gene regulation and lens development

The vertebrate lens is a tissue composed of terminally differentiated fiber cells and anterior lens epithelial cells. The abundant, preferential expression of the soluble proteins called crystallins creates a transparent, refractive index gradient in the lens. Several transcription factors such as Pax6, Sox1, and L-Maf have been shown to regulate lens development. Here we show that mice lacking the transcription factor c-Maf are microphthalmic secondary to defective lens formation, specifically from the failure of posterior lens fiber elongation. The marked impairment of crystallin gene expression observed is likely explained by the ability of c-Maf to transactivate the crystallin gene promoter. Thus, c-Maf is required for the differentiation of the vertebrate lens.

Characterization of the Saccharomyces cerevisiae ERG27 gene encoding the 3-keto reductase involved in C-4 sterol demethylation

The last unidentified gene encoding an enzyme involved in ergosterol biosynthesis in Saccharomyces cerevisiae has been cloned. This gene, designated ERG27, encodes the 3-keto sterol reductase, which, in concert with the C-4 sterol methyloxidase (ERG25) and the C-3 sterol dehydrogenase (ERG26), catalyzes the sequential removal of the two methyl groups at the sterol C-4 position. We developed a strategy to isolate a mutant deficient in converting 3-keto to 3-hydroxy-sterols. An ergosterol auxotroph unable to synthesize sterol or grow without sterol supplementation was mutagenized. Colonies were then selected that were nystatin-resistant in the presence of 3-ketoergostadiene and cholesterol. A new ergosterol auxotroph unable to grow on 3-ketosterols without the addition of cholesterol was isolated. The gene (YLR100w) was identified by complementation. Segregants containing the YLR100w disruption failed to grow on various types of 3-keto sterol substrates. Surprisingly, when erg27 was grown on cholesterol- or ergosterol-supplemented media, the endogenous compounds that accumulated were noncyclic sterol intermediates (squalene, squalene epoxide, and squalene dioxide), and there was little or no accumulation of lanosterol or 3-ketosterols. Feeding experiments in which erg27 strains were supplemented with lanosterol (an upstream intermediate of the C-4 demethylation process) and cholesterol (an end-product sterol) demonstrated accumulation of four types of 3-keto sterols identified by GC/MS and chromatographic properties: 4-methyl-zymosterone, zymosterone, 4-methyl-fecosterone, and ergosta-7,24 (28)-dien-3-one. In addition, a fifth intermediate was isolated and identified by 1H NMR as a 4-methyl-24,25-epoxy-cholesta-7-en-3-one. Implications of these results are discussed.

The interferon-inducible murine p48 (ISGF3γ) gene is regulated by protooncogene c-myc

p48 protein is an integral component of the multimeric interferon (IFN)-regulated transcription factor, ISGF3. We have shown earlier that this gene is regulated by a novel IFN-γ-regulated element. In addition to the IFN-regulated element, a myc–max binding site is also present in this promoter. In this investigation we have studied the role of this site in the regulation of the p48 gene. In serum-induced quiescent cells Myc up-regulated the expression of p48 mRNA. We show that the protooncogene Myc regulates the expression of p48 through the element CACGTG. Mutations in this motif abolish Myc-inducibility of the reporter genes carrying p48 promoter elements. Purified Myc and Max proteins interact with the Myc-stimulated element of the p48 promoter. We also show that cells lacking p48 expression are highly susceptible to the cytocidal action of anticancer drugs. Taken together these data suggest that p48 may function as an anti-stress cell survival factor.

Inactivation of the α C protein antigen gene, bca, by a novel shuttle/suicide vector results in attenuation of virulence and immunity in group B Streptococcus

The α C protein of group B Streptococcus (GBS) is a major surface-associated antigen. Although its role in the biology and virulence of GBS has not been defined, it is opsonic and capable of eliciting protective immunity. The α C protein is widely distributed among clinical isolates and is a potential protein carrier and antigen in conjugate vaccines to prevent GBS infections. The structural gene for the α C protein, bca, has been cloned and sequenced. The protein encoded by bca is related to a class of surface-associated proteins of Gram-positive cocci involved in virulence and immunity. To investigate the potential roles of the α C protein, bca null mutants were generated in which the bca gene was replaced with a kanamycin resistance cassette via homologous recombination using a novel shuttle/suicide vector. Studies of lethality in neonatal mice showed that the virulence of the bca null mutants was attenuated 5- to 7-fold when compared with the isogenic wild-type strain A909. Significant differences in mortality occurred in the first 24 h, suggesting that the role of the α antigen is important in the initial stages of the infection. In contrast to A909, bca mutants were no longer killed by polymorphonuclear leukocytes in the presence of α-specific antibodies in an in vitro opsonophagocytic assay. In contrast to previous studies, α antigen expression does not appear to play a role in resistance to opsonophagocytosis in the absence of α-specific antibodies. In addition, antibodies to the α C protein did not passively protect neonatal mice from lethal challenge with bca mutants, suggesting that these epitopes are uniquely present within the α antigen as expressed from the bca gene. Therefore, the α C protein is important in the pathogenesis of GBS infection and is a target for protective immunity in the development of GBS vaccines.