Gene Mutations and Polymorphisms of TP53 and FHIT in Chronic Esophagitis and Esophageal Carcinoma

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

The Xmnl polymorphic site 5 ' to the gene G gamma in a Brazilian patient with sickle cell anaemia - fetal haemoglobin concentration, haematology and clinical features

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Haplotype effects on matrix metalloproteinase-1 gene promoter activity in cancer cells

Increased expression of matrix metalloproteinase-1 (MMP1) is associated with poor prognosis in cancers. Several single nucleotide polymorphisms (-1607GG > G, -839G > A, -755G > T, -519A > G, -422T > A, -340C > T, and 320C > T) in the MMP1 gene promoter have recently been identified. In this study, we assessed the functional effects of these polymorphisms on MMP1 gene promoter activity in cell lines of melanoma (A2058 and A375), breast cancer (MCF7 and MDA-MB-231), lung cancer (A549 and H69), and colorectal cancer (HT-29, SW-620) by comparing the promoter strengths of 10 most common haplotypes deriving from these polymorphisms. In A2058 cells, the GG-G-G-A-T-T-T and GG-G-G-A-C-T haplotypes had 2-fold higher promoter activity than the GG-G-T-A-T-T-C, GG-G-G-A-A-T-T, GG-G-G-A-T-T-C, and GG-G-G-A-A-C-T haplotypes, which in turn, had 3-fold higher promoter activity than the G-G-T-A-A-C-T, G-A-T-G-T-T-T, G-A-T-G-A-C-T, and G-A-T-G-A-T-G haplotypes. In A375 and MDA-MB-231 cells, high expression haplotypes include not only the -1607GG-bearing haplotypes but also the G-A-T-G-A-T-T haplotype containing the -1607G allele. A similar trend was detected in A549 cells. In addition, in A549 cells, the GG-G-G-A-T-T-T haplotype had > 2-fold higher promoter activity than several other 1607GG-bearing haplotypes. In MCF7 cells, the GG-G-G-A-T-T-T and G-G-T-A-A-C-T haplotypes had 1.5- to 4-fold higher promoter activity than the other haplotypes. These results suggest that the polymorphisms exert haplotype effects on the transcriptional regulation of the MMP1 gene in cancer cells, and indicate a need to examine haplotypes rather than any single polymorphism in genetic epidemiologic studies of the MMP1 gene in cancers.

Association of CSSM066 and ILSTS011 microsatellite markers and thyroglobulin gene SNP with backfat in Canchim cattle

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

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.

A novel PCR-RFLP assay for the detection of the single nucleotide polymorphism at position+1440 in the human CXCR2 gene

We designed a novel PCR-RFLP assay to genotype for the CXCR2 +1440 (G/A) single nucleotide polymorphism, which provides a simple, low-cost, practical, and reproducible method. Allele frequencies in healthy Brazilian individuals were found to be 0.65% for allele A and 0.35% for allele G.

Hemoglobin and HFE gene polymorphisms in Brazilian populations in regions endemic for malaria

Our objective was to determine how the distribution of red blood cell diseases is related to malaria occurrence in north Brazil, a region endemic for malaria. We evaluated the incidence of two mutations in the HFE gene, H63D and C282Y, in two study groups: a control blood donor group, with no indication of malaria infection, and a group constituted of malaria patients of four states of the Amazonian region. The hemoglobin polymorphisms were obtained by HPLC and classical laboratory methodologies, and the two mutations in the HFE gene were assayed by PCR-RFLP. We found a high frequency of alpha thalassemia, but there were no significant differences between blood donors and malaria patients. There were also no significant differences in the frequencies of HbA(2); however, the frequency of HbF was significantly different in individuals with malaria from Para and Rondonia. The mean number of reticulocytes was significantly reduced in the blood donors from the northern region, suggesting an adaptive strategy of these populations to parasitic attack by Plasmodium. Most individuals were heterozygous for the H63D allele of the HFE gene in both study groups. In the blood donors group, the greatest frequency of the H63D allele was found in Caucasians of all the states. In the malaria patients group in Rondonia, there was a high frequency of the H63D allele among the non-Caucasians. In the other states, and in the malaria patients group, the H63D allele was the most frequent among the Caucasians. Based on our results, we suggest that the maintenance of polymorphism of the mutations in the gene HFE can be explained by selective factors other than malaria, or it is due to simple allelic oscillation and by the constant gene flow among the populations in Brazil.

Use of PCR-RFLP (Polymerase Chain Reaction-Restricted Fragment Length Polymorphism) in the gene of the enzyme Stearoyl-CoA-Desaturase in Bubalus bubalis

The milk is an important food because it contents Conjugated Linoleic Acids (CIA). These fatty acids are synthesized in mammary gland under action of the enzyme Stearoyl CoA-Desaturase (SCD) and have showed some positive effects in human disease prevention and treatments. A variation of CLA in milk fat exists and can be partially explained by the different levels of expression of SCD. The aim was to study part of the encoding regions of SCD's gene using PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism). Genomic DNA was extracted from lactating Murrah females. After this, PCR reactions were made by using primers Z (sic) (sic) D1 that encloses exon I, II and intron I. The fragments amplified are composed by 938 pb. Then, RFLP techniques were applied in the fragments using the restriction enzymes Pst I and Sma I. The enzyme Pst I has generated fragments of 788pb and 150bp and the Sma I has generated fragments of 693pb and 245pb. All the animals showed the same migration standard for both enzymes, characterizing a genetic monomorphism for this region of SCD gene. The analysis determined that there aren't genetic differences between these animals in the studied regions by using Pst I and Sma I enzymes.

Identification of polymorphisms in the 5 '-untranslated region of the TAFI gene: relationship with plasma TAFI levels and risk of venous thrombosis

Background and Objectives. Thrombin activatable fibrinolysis inhibitor (TAFI) plays an important role in hemostasis, functioning as a potent fibrinolysis inhibitor. TAFI gene variations may contribute to plasma TAFI levels and thrombotic risk.Design and Methods. We sequenced a 2083-bp region of the 5 ' -regulatory region of the TAFI gene in 127 healthy subjects searching for variations, and correlated identified polymorphisms with plasma TAFI levels. TAFI polymorphisms were examined as risk factors for venous thrombosis by determining their prevalence in 388 patients with deep venous thrombosis (DVT) and in 388 controls.Results. Seven novel polymorphisms were identified: -152 A/G, -438 A/G, -530 C/T, -1053 T/C, -1102 T/G, -1690 G/A, and -1925 T/C. -152 A/G, -530 C/T and -1925 T/C were found to be in strong linkage disequilibrium, as were the -438 A/G, -1053 T/C, -1102 T/G and -1690 G/A, Plasma TAFI levels were higher in -43866/-1053CC/-1102GG/-1690AA homozygotes than In -438AG/-1053TC/-1102TG/-1690GA heterozygotes, and -438AA/-1053TT/-1102TT/-1690GG homozygotes had the lowest TAFI levels (p=0.0003). TAFI concentrations in -152AA/-530CC/-1925TT homozygotes were somewhat higher but not significantly different from levels observed for -152AG/-530CT/-1925TC heterozygotes, Taken in combination, -438AG/-1053TC/-1102TG/-1690GA and -438AA/-1053TT/-1102TT/-1690GG yielded an OR for DVT of 0.8 (95%CI: 0.6-1). in subjects aged < 35 years the OR was 0.7 (95%CI: 0.5-1.1), the OR for -152AG/-530CT/-1925TC was 1 (95%CI: 0.5-2.2) in the whole group of patients and controls, whereas in subjects aged <35 years the OR was 0.1 (95%CI: 0.02-0.9).Interpretation and Conclusions. Polymorphisms in the TAFI promoter determine plasma antigen levels and may influence the risk of venous thrombophilia. <(c)>2001, Ferrata Storti Foundation.

Interferon-gamma+874 Polymorphism in the First Intron of the Human Interferon-gamma Gene and Kidney Allograft Outcome

Background. Despite advances in immunosuppressive therapy in the past decade, allograft rejection remains an important cause of kidney graft failure. Cytokines play a major role in the inflammatory and immune responses that mediate allograft outcomes. Several studies have shown that the production of cytokines varies among individuals. These variations are determined by genetic polymorphisms, most commonly within the regulatory region of cytokine genes. The aim of the present study was to assess the effect of allelic variation on acute rejection episodes (ARE) or chronic allograft nephropathy (CAN) after kidney transplantation.Methods. To determine a possible correlation between the interferon (INF)-gamma +874 polymorphism and kidney allograft outcome, we isolated genomic DNA from 74 patients who underwent isolated kidney allografts and were classified into 2 groups-a rejection and a nonrejection group-for comparison with a control group of 163 healthy subjects.Results. We genotyped INF-gamma +874 polymorphisms in all groups. The transplant group showed a significantly increased homozygous genotype T/T (P = .0118) compared with healthy controls. Similarly, considering only patients with CAN, the homozygous genotype T/T (P = .0067) was significantly increased compared with the healthy controls. The rejection group indicated a significant increased homozygous genotype Tic compared with the control group (P = .0061).Conclusion. Homozygous genotype T/T was associated with increased levels of INF-gamma and greater numbers among the rejection and CAN cohorts.

IFNG +874 T > A single nucleotide polymorphism is associated with leprosy among Brazilians

Leprosy is a chronic infectious disease caused by Mycobacterium leprae, a low virulence mycobacterium, and the outcome of disease is dependent on the host genetics for either susceptibility per se or severity. The IFNG gene codes for interferon-gamma (IFN-gamma), a cytokine that plays a key role in host defense against intracellular pathogens. Indeed, single nucleotide polymorphisms (SNPs) in IFNG have been evaluated in several genetic epidemiological studies, and the SNP +874T > A, the +874T allele, more specifically, has been associated with protection against infectious diseases, especially tuberculosis. Here, we evaluated the association of the IFNG locus with leprosy enrolling 2,125 Brazilian subjects. First, we conducted a case-control study with subjects recruited from the state of So Paulo, using the +874 T > A (rs2430561), +2109 A > G (rs1861494) and rs2069727 SNPs. Then, a second study including 1,370 individuals from Rio de Janeiro was conducted. Results of the case-control studies have shown a protective effect for +874T carriers (OR(adjusted) = 0.75; p = 0.005 for both studies combined), which was corroborated when these studies were compared with literature data. No association was found between the SNP +874T > A and the quantitative Mitsuda response. Nevertheless, the spontaneous IFN-gamma release by peripheral blood mononuclear cells was higher among +874T carriers. The results shown here along with a previously reported meta-analysis of tuberculosis studies indicate that the SNP +874T > A plays a role in resistance to mycobacterial diseases.

Determination of neutral polymorphisms (frameworks) of the human beta globin gene in beta thalassaemias by PCR/DGGE

Purpose: Considering the importance of type beta thalassaemias as hereditary syndromes of high significance in different populations of Mediterranean origin and, by extension, in the Brazilian population, the objective of the present study was to determine by PCR/DGGE the gene structures responsible for neutral polymorphisms (frameworks) observed in the human beta globin gene associated with the mutations responsible for type beta thalassaemias in a sample of the Brazilian population and, more specifically, of the population of the State of São Paulo. Patients and methods: Thirty individuals with beta thalassaemic mutations were analyzed: 22 mutations were in codon 39 (C->T), 5 in IVS1-110 (G->A), 2 in IVS1-6 (T->C) and 1 in IVS1-1 (G->A). DNA was extracted and selective amplification was performed by PCR extending from position IVS1 nt 46 to IVS2 nt 126 (474 pb). The product was then analyzed by polyacrylamide gel electrophoresis on a denaturing 10-60% urea/formamide gradient. Results: The results demonstrated that, as expected, the mutations responsible for type beta thalassaemia observed in this population are of Mediterranean origin, with 73% distribution represented by codon 39,17% by IVS1-110, 7% by IVS1-6 and 3% by IVS1-1. In turn, framework distribution seems to indicate a higher frequency of Fr 1-1 in codon 39 and IVS1-110, of Fr 1-3 in IVS1-6 and of Fr 1-2 in IVS1-1. Conclusions: These results permit us to conclude that gene amplification by PCR followed by DGGE is an appropriate method for the separation of DNA molecules that differ even by a single base change and therefore can be utilized to detect the alterations observed in the human beta globin gene. This methodology shows that, using only a pair of primers, it is possible to define the frameworks that are observed in the beta globin gene.

Effects of CSN3 and LGB gene polymorphisms on production traits in beef cattle

The objective of the present study was to estimate the allele and genotype frequencies of the CSN3/Hinfl and LGB/HaeIII gene polymorphisms in beef cattle belonging to different genetic groups, and to determine the effects of these polymorphisms on growth and carcass traits in these animals, which are submitted to an intensive production model. Genotyping was performed on 79 Nelore, 30 Canchim (5/8 Charolais + 3/8 Zebu) and 275 crossbred cattle originating from the crosses of Simmental (n = 30) and Angus (n = 245) sires with Nelore females. Body weight, weight gain, dressing percentage, longissimus dorsi area and backfat thickness were fitted using the GLM procedure, and least square means of the genotypes were compared by the F test. The results showed that the CSN3/Hinfl and LGB/HaeIII polymorphisms did not have any effect on growth or carcass traits (p > 0.05). Copyright by the Brazilian Society of genetics.