Molecular analysis of the Von Hippel-Lindau (VHL) gene in a family with non-syndromic pheochromocytoma: the importance of genetic testing

Dois pacientes índices da família analisada neste estudo foram submetidos a adrenalectomia bilateral devido a feocromocitoma. Foi, então, realizado o estudo genético dos pacientes e de sete parentes de primeiro grau. Os dois pacientes com feocromocitoma e dois outros membros assintomáticos da família apresentaram a mutação c496G>T no exon 3 do gene VHL. A família perdeu seguimento médico. Três anos após a realização da avaliação genética, a irmã dos pacientes, portadora da mutação, foi encaminhada para o nosso serviço após uma gestação complicada por pré-eclampsia. Ela referia paroxismos sugestivos de feocromocitoma, mas as metanefrinas urinárias eram negativas. Entretanto, a tomografia computadorizada de abdômen evidenciou uma massa adrenal que também se contrastou na cintilografia com metaiodobenzilguanidina (MIBG). Esse estudo mostra que a análise molecular do paciente índice pode levar à identificação de parentes assintomáticos portadores da mutação. Além disso, mesmo com as metanefrinas urinárias negativas, a identificação de uma mutação específica levou a um aumento da suspeita e detecção de feocromocitoma na irmã dos afetados pela doença.

p53 gene analysis in childhood B non - Hodgkin's lymphoma

CONTEXTO: Alterações do gene supressor de tumor p53, como mutações e deleções, são lesões genéticas encontradas com maior freqüência nas neoplasias humanas, incluindo câncer de mama, pulmão e cólon. Entre as malignidades hematológicas, o gene 53 é freqüentemente mutado no linfoma de Burkitt, sendo detectadas mutações em 30-40% das amostras tumorais e em 70% das linhagens celulares. OBJETIVO: Analisar as alterações do gene p53 em crianças com linfoma não-Hodgkin de origem B. TIPO DE ESTUDO: Estudo descritivo. LOCAL: Centro de Oncologia Terciário. PARTICIPANTES: O estudo analisou 12 pacientes com linfoma não-Hodgkin B classificados como linfoma de Burkitt. A análise de possíveis mutações do gene p53 foi realizada pela técnica de PCR-SSCP dos exons 5, 6 ,7 e 8/9 do gene. RESULTADOS: Um padrão anormal de migração foi observado em quatro pacientes (33.3%), em um paciente no exon 6 e em três no exon 7. Os casos positivos incluíam dois pacientes que evoluíram para o óbito por progressão da doença. CONCLUSÃO: Esses resultados preliminares sugerem que as alterações do gene p53 são freqüentes em crianças com linfoma de Burkitt e podem contribuir para patogênese ou progressão da doença.

Analysis of CDKN1A polymorphisms: markers of cancer susceptibility?

The CDKN1A (TP21)(2) gene encodes a 21-kD protein that is a critical downstream mediator of wild-type TP53 and an important regulator of the cell cycle. Failure in the function of this gene would be expected to result in abnormal cell proliferation and transformation. Tumor-associated mutations of the coding region of the TP21 are rare. on the other hand, some TP21 polymorphisms have been identified and characterized by single base substitutions. In the present study, we investigated the potential role of TP21 gene polymorphisms in skin, head, and neck tumorigenesis. A total of 261 samples were examined by polymerase chain reaction single-strand conformational analysis, and one mutation at codon 31 and four polymorphisms in exons 2 (codon 55) and 3 [nucleotide (nt)590] and in promoter region (nt2298) were identified. In conclusion, this investigation confirmed the rarity of mutations in this gene, arguing against a role for TP21 mutations in skin, head, and neck cancers. Also, our results show significant differences in nt2298 allele frequencies between normal individuals and skin malignant tumors (P < 0.05). The results suggest that this polymorphism affects TP21 transactivator binding and may be important during the pathogenesis of skin cancer. (C) 2003 Elsevier B.V. All rights reserved.

Cytotoxicity, genotoxicity and antimutagenicity of hexane extracts of Agaricus blazei determined in vitro by the comet assay and CHO/HGPRT gene mutation assay

Agaricus blazei Murrill ss. Heinem, known as the sun mushroom or himematsutake, is a basidiomycete native to Brazil, which is popular for its medicinal properties. The aim of this study was to test hexane extracts (one fraction and its four sub-fractions) of A. blazei for bioactivity in cultured mammalian cells (CHO-K1). The comet assay, the colony forming assay (CFA) and CHO/HGPRT gene mutation assay were used respectively to determine genotoxicity, cytotoxicity and antimutagenicity of these extracts at different concentrations. The cells were incubated in culture medium and treated for 3 h according to the standard protocol for each assay. The DNA damage-inducing agent ethylmethane sulfonate (EMS) was utilized as the positive control and also in combination with extracts to test for a protective effect. Statistical analysis of the data was performed using analysis of variance (ANOVA) and Tukey's test. A relationship between cytotoxicity and genotoxicity could be established and two extracts EH6B and EH6D showed a protective tendency, while the others did not, with the primary extract EH6 causing the most substantial damage to genetic material. These findings warrant more in-depth studies of the active principles of this mushroom. (c) 2005 Elsevier Ltd. All rights reserved.

Gastric adenocarcinoma and Helicobacter pylori: Correlation with p53 mutation and p27 immunoexpression

Introduction: Helicobacter pylori infection is an established risk factor for gastric cancer development, but the exact underlying mechanism still remains obscure. The inactivation of tumor suppressor genes such as p53 and p27(KIP1) is a hypothesized mechanism, although there is no consensus regarding the influence of H. pylori cagA(+) in the development of these genetic alterations. Goals: To verify the relationship among H. pylori infection, p53 mutations and p27(Kip1) Protein (p27) expression in gastric adenocarcinomas (GA) seventy-four tissues were assayed by PCR for H. pylori and cagA presence. Mutational analysis of p53 gene was performed by single-strand conformation polymorphism (SSCP). Seventy tissues were analyzed by an immunohistochemical method for p27 expression. Results: From the samples examined, 95% (70/74) were H. pylori positive, 63% cagA(+). Altered p53 electrophoretic mobility was found in 72% of cases and significantly more frequent in the presence of cagA. Considerable reduction in p27 expression (19%) was found with a tendency for association between cagA(+) and p27(-), although the results were not statistically significant. Concomitant alterations of both suppressor genes were detected in 60% of cases. In the cases cagA(+), 66.7% of them had these concomitant alterations. Conclusions: The data suggest that H. pylori cagA(+) contributes to p53 alteration and indicate that concomitant gene inactivation, with reduced p27 expression, may be a mechanism in which H. pylori can promote the development and progression of gastric cancer. (C) 2010 Elsevier Ltd. All rights reserved.

The frequency of 844ins68 mutation in the cystathionine β-synthase gene is not increased in patients with venous thrombosis

Background and Objectives. A frequent mutation in the cystathionine β- synthase (CBS) gene (844ins68, a 68-bp insertion in the coding region of exon 8) was recently discovered. In the present study we investigated this mutation as a candidate risk factor for venous thrombosis. Design and Methods. The prevalence of the 844ins68 CBS mutation was determined in 101 patients with objectively diagnosed deep venous thrombosis and in 101 healthy controls matched for age, sex and race. PCR amplification of a DNA fragment containing exon 8 of the CBS gene was employed to determine the genotypes. Additionally, Bsrl restriction enzyme digestion of the PCR products was performed in all samples from carriers of the insertion, to test for concurrent presence of a second mutation (T833C) in the CBS gene. Results. The insertion was found in 21 out of 101 patients (20.8%; allele frequency 0.109) and in 20 out of 101 controls (19.8%; allele frequency 0.114), yielding a relative risk for venous thrombosis related to the 844ins68 CBS mutation close to 1.0. In addition, the T833C CBS mutation was detected in all alleles carrying the 844ins68 CBS insertion, confirming the co- inheritance of the two mutations. Interpretation and Conclusions. Our findings do not support the hypothesis that the 844ins68 mutation in the CBS gene is a genetic risk factor for venous thrombosis.

Kras oncogene analysis of non-melanoma skin tumors in Southeastern Brazil

Skin cancers are the most common human malignant neoplasia and their incidence is growing, chiefly in tropical countries. There is evidence that ultraviolet (UV) radiation present in sunlight is important for genetic damage. Mutations due to such damage could be responsible for alterations in oncogenes and tumor suppressor genes. Recent studies have reported remarkable differences in mutation frequency of the RAS proto-oncogene in non-melanoma skin cancers. These findings may reflect differences in the molecular epidemiology of cutaneous tumors found in geographical areas with diverse sun exposure and ethnical origins of their populations. Our study proposed to perform molecular analyses of skin tumors on patients living in southeastern Brazil, in areas with high levels of sun exposure. DNA from eight solar keratose (SK), 26 basal cell carcinomas (BCC) and 19 squamous cell carcinomas (SCC) was submitted to PCR-SSCP analysis for codons 12, 13 and 61. Contradicting other authors, we found no mutations in codons 12,13 but detected two BCCs and one SCC with a mutation in codon 61. These findings suggest that the activation of KRAS oncogene may contribute to the pathogenicity of cutaneous lesions in southeastern Brazil.

Analysis of agonist and antagonist effects on thyroid hormone receptor conformation by hydrogen/deuterium exchange

Thyroid hormone receptors (TRs) are ligand-gated transcription factors with critical roles in development and metabolism. Although x-ray structures of TR ligand-binding domains (LBDs) with agonists are available, comparable structures without ligand (apo-TR) or with antagonists are not. It remains important to understand apo-LBD conformation and the way that it rearranges with ligands to develop better TR pharmaceuticals. In this study, we conducted hydrogen/deuterium exchange on TR LBDs with or without agonist (T 3) or antagonist (NH3). Both ligands reduce deuterium incorporation into LBD amide hydrogens, implying tighter overall folding of the domain. As predicted, mass spectroscopic analysis of individual proteolytic peptides after hydrogen/ deuterium exchange reveals that ligand increases the degree of solvent protection of regions close to the buried ligand-binding pocket. However, there is also extensive ligand protection of other regions, including the dimer surface at H10-H11, providing evidence for allosteric communication between the ligand-binding pocket and distant interaction surfaces. Surprisingly, Cterminal activation helix H12, which is known to alter position with ligand, remains relatively protected from solvent in all conditions suggesting that it is packed against the LBD irrespective of the presence or type of ligand. T 3, but not NH3, increases accessibility of the upper part of H3-H5 to solvent, and we propose that TR H12 interacts with this region in apo-TR and that this interaction is blocked by T 3 but not NH3.Wepresent data from site-directed mutagenesis experiments and molecular dynamics simulations that lend support to this structural model of apo-TR and its ligand-dependent conformational changes. (Molecular Endocrinology 25: 15-31, 2011). Copyright © 2011 by The Endocrine Society.

Genome-Wide Analysis Reveals Selection for Important Traits in Domestic Horse Breeds

Intense selective pressures applied over short evolutionary time have resulted in homogeneity within, but substantial variation among, horse breeds. Utilizing this population structure, 744 individuals from 33 breeds, and a 54,000 SNP genotyping array, breed-specific targets of selection were identified using an FST-based statistic calculated in 500-kb windows across the genome. A 5.5-Mb region of ECA18, in which the myostatin (MSTN) gene was centered, contained the highest signature of selection in both the Paint and Quarter Horse. Gene sequencing and histological analysis of gluteal muscle biopsies showed a promoter variant and intronic SNP of MSTN were each significantly associated with higher Type 2B and lower Type 1 muscle fiber proportions in the Quarter Horse, demonstrating a functional consequence of selection at this locus. Signatures of selection on ECA23 in all gaited breeds in the sample led to the identification of a shared, 186-kb haplotype including two doublesex related mab transcription factor genes (DMRT2 and 3). The recent identification of a DMRT3 mutation within this haplotype, which appears necessary for the ability to perform alternative gaits, provides further evidence for selection at this locus. Finally, putative loci for the determination of size were identified in the draft breeds and the Miniature horse on ECA11, as well as when signatures of selection surrounding candidate genes at other loci were examined. This work provides further evidence of the importance of MSTN in racing breeds, provides strong evidence for selection upon gait and size, and illustrates the potential for population-based techniques to find genomic regions driving important phenotypes in the modern horse. © 2013 Petersen et al.

MODY 2: Mutation identification and molecular ancestry in a Brazilian family

Maturity Onset Diabetes of the Young (MODY) is a heterogeneous group of genetic diseases characterized by a primary defect in insulin secretion and hyperglycemia, non-ketotic disease, monogenic autosomal dominant mode of inheritance, age at onset less than 25. years, and lack of auto-antibodies. It accounts for 2-5% of all cases of non-type 1 diabetes. MODY subtype 2 is caused by mutations in the glucokinase (GCK) gene. In this study, we sequenced the GCK gene of two volunteers with clinical diagnosis for MODY2 and we were able to identify four mutations including one for a premature stop codon (c.76C>T). Based on these results, we have developed a specific PCR-RFLP assay to detect this mutation and tested 122 related volunteers from the same family. This mutation in the GCK gene was detected in 21 additional subjects who also had the clinical features of this genetic disease. In conclusion, we identified new GCK gene mutations in a Brazilian family of Italian descendance, with one due to a premature stop codon located in the second exon of the gene. We also developed a specific assay that is fast, cheap and reliable to detect this mutation. Finally, we built a molecular ancestry model based on our results for the migration of individuals carrying this genetic mutation from Northern Italy to Brazil. © 2012 Elsevier B.V.

Analysis of HCV quasispecies dynamic under selective pressure of combined therapy

Background: The quasispecies composition of Hepatitis C virus (HCV) could have important implications with regard to viral persistence and response to interferon-based therapy. The complete NS5A was analyzed to evaluate whether the composition of NS5A quasispecies of HCV 1a/1b is related to responsiveness to combined interferon pegylated (PEG-IFN) and ribavirin therapy.Methods: Viral RNA was isolated from serum samples collected before, during and after treatment from virological sustained responder (SVR), non-responder (NR) and the end-of-treatment responder patients (ETR). NS5A region was amplified, cloned and sequenced. Six hundred and ninety full-length NS5A sequences were analyzed.Results: This study provides evidence that lower nucleotide diversity of the NS5A region pre-therapy is associated with viral clearance. Analysis of samples of NRs and the ETRs time points showed that genetic diversity of populations tend to decrease over time. Post-therapy population of ETRs presented higher genetic distance from baseline probably due to the bottleneck phenomenon observed for those patients in the end of treatment. The viral effective population of those patients also showed a strong decrease after therapy. Otherwise, NRs demonstrated a continuous variation or stability of effective populations and genetic diversity over time that did not seem to be related to therapy. Phylogenetic relationships concerning complete NS5A sequences obtained from patients did not demonstrate clustering associated with specific response patterns. However, distinctive clustering of pre/post-therapy sequences was observed. In addition, the evolution of quasispecies over time was subjected to purifying or relaxed purifying selection. Codons 157 (P03), 182 and 440 (P42), 62 and 404 (P44) were found to be under positive selective pressure but it failed to be related to the therapy.Conclusion: These results confirm the hypothesis that a relationship exists between NS5A heterogeneity and response to therapy in patients infected with chronic hepatitis C. © 2013 Jardim et al.; licensee BioMed Central Ltd.

Nucleotide and phylogenetic analysis of human papillomavirus types 6 and 11 isolated from recurrent respiratory papillomatosis in Brazil

There are few studies about the distribution of natural molecular variants of low-risk HPVs. Our aim was to evaluate the E6 early gene variability among HPV-6 and HPV-11 isolates detected in recurrent respiratory papillomatosis (RRP) samples obtained in a cohort of Brazilian patients. We also performed a phylogenetic analysis in order to compare nucleotide sequences identified in our study with previously reported isolates from different anatomic sites (laryngeal papillomas, genital warts, cervical cancer and anal swabs) obtained from other parts of the world to determine the phylogenetic relationships of variants detected in Brazil. The complete coding region of the E6 gene of 25 samples was cloned and sequenced: 18 isolates of HPV-6 (72%) and 7 isolates of HPV-11 (28%). A total of four different HPV-6 genomic variants and two HPV-11 genomic variants was identified. It was not possible to correlate specific variants with disease severity. Phylogenetic trees for both HPV types were constructed enclosing both E6 sequences detected in our study and formerly published sequences. In both phylogenetic trees, the sequences from Brazil did not group together. We could not establish a geographical association between HPV-6 or HPV-11 variants, unlike HPV-16 and HPV-18. © 2013 Elsevier B.V.

Allele frequency of hereditary equine regional dermal asthenia in American Quarter horses in Brazil determined by quantitative real-time PCR with high resolution melting analysis

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

Diagnostic tests for hepatitis C: Recent trends in electrochemical immunosensor and genosensor analysis

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

Impact of FLT3 Internal Tandem Duplication on the Outcome of Related and Unrelated Hematopoietic Transplantation for Adult Acute Myeloid Leukemia in First Remission: A Retrospective Analysis

Purpose Patients with acute myeloid leukemia (AML) and FLT3/internal tandem duplication (FLT3/ITD) have poor prognosis if treated with chemotherapy only. Whether this alteration also affects outcome after allogeneic hematopoietic stem-cell transplantation (HSCT) remains uncertain. Patients and Methods We analyzed 206 patients who underwent HLA-identical sibling and matched unrelated HSCTs reported to the European Group for Blood and Marrow Transplantation with a diagnosis of AML with normal cytogenetics and data on FLT3/ITD (present: n = 120, 58%; absent: n = 86, 42%). Transplantations were performed in first complete remission (CR) after myeloablative conditioning. Results Compared with FLT3/ITD-negative patients, FLT3/ITD-positive patients had higher median leukocyte count at diagnosis (59 v 21 x 10(9)/L; P < .001) and shorter interval from CR to transplantation (87 v 99 days; P = .04). Other characteristics were similar in the two groups. At 2 years, relapse incidence (RI; +/- standard deviation) was higher (30% +/- 5% v 16% +/- 5%; P = .006) and leukemia-free survival (LFS) lower (58% +/- 5% v 71% +/- 6%; P = .04) in FLT3/ITD-positive compared with FLT3/ITD-negative patients. In multivariate analyses, FLT3/ITD led to increased RI (hazard ratio [HR], 3.4; 95% CI, 1.46 to 7.94; P = .005), as did older age, female sex, shorter interval between CR and transplantation, and higher number of chemotherapy courses before achieving CR. FLT3/ITD positivity was associated with decreased LFS (HR, 0.37; 95% CI, 0.19 to 0.73; P = .002), along with older age and higher number of chemotherapy courses before achieving CR. Conclusion FLT3/ITD adversely affected the outcome of HSCT in the same direction it does after chemotherapy; despite this, more than half of the patients harboring this mutation who received transplants were alive and leukemia free at 2 years. To further improve the results, use of FLT3 inhibitors before or after HSCT deserves investigation.